1. science
2. medicine
3. genetics

Alma Mater Studiorum – Università di Bologna
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Dottorato di Ricerca in Biologia Cellulare Molecolare
Ciclo XXVI
Settore Concorsuale di afferenza: 05/I1 Genetica e Microbiologia
Settore Scientifico disciplinare: BIO/18 Genetica
Drosophila melanogaster as a model to study
host-parasitoid interactions:
the case of the polydnaviral protein TnBVANK1
Presentata da: Luca VALZANIA
Coordinatore dottorato
Relatore
Prof. Vincenzo SCARLATO
Prof. Giuseppe GARGIULO
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Esame finale anno 2014
_________________________________________________________Table of contents
Abstract
4
1-Introduction
6
1.1 Parasitoids
8
1.2 Polydnaviruses
10
1.3 The host-parasitoid association Heliothis virescensToxoneuron nigriceps
12
1.4 The Braconidae polydnavirus associated with T. nigriceps
(TnBV)
14
1.5 TnBVank1
17
1.6 Drosophila melanogaster as a model system
18
1.7 Overview of Drosophila development
19
1.8 The role of the steroid hormone ecdysone in Drosophila
melanogaster
21
1.9 The prothoracic gland: the site of ecdysteroidogenesis
28
1.10 Ecdysone biosynthesis
29
1.11 Cholesterol trafficking in steroidogenic cells
33
2-Research Aims
36
3-Materials and Methods
38
3.1 Fly food
39
3.2 Fly strains
39
3.3 Genetic crosses
41
3.4 Larval length measurements
42
3.5 20-hydroxyecdysone (20E) titration
42
3.6 Rescue experiments
42
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3.7 Prothoracic gland and cellular size measurements
43
3.8 Immunofluorescence Microscopy
43
3.9 Antibodies
44
3.10 Terminal deoxynucleotidyl transferase-mediated
dUTP Nick End Labeling (TUNEL) Analysis
46
3.11 Filipin and Oil Red O stainings
46
3.12 Colocalization analysis
47
3.13 Statistical analyses
47
4-Results
48
4.1 The expression of TnBVank1 arrests development
during larval stage three
49
4.2 TnBVank1 expression in the prothoracic gland cells
blocks the larva-to-pupa transition
52
4.3 phm-Gal4>TnBVank1 larvae contain low levels of 20hydroxyecdysone
54
4.4 The expression of TnBVank1 affects the PG
morphology
58
4.5 The expression of TnBVank1 in the PG impairs the
cytoskeletal network
64
4.6 TnBVank1 expression causes increased accumulation of
lipids in PG cells
68
4.7 The organization of the cholesterol trafficking pathway
in PG cells
70
4.8 The endocytic pathway is altered in PG cells expressing
TnBVank1
72
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4.9 TnBVANK1 is localized in multivesicular bodies
74
4.10 The role of ALIX in the endosomal trafficking
76
4.11 In PG cells TnBVANK1 colocalizes with ALIX
positive endosomes
77
4.12 ALIX knockdown in the PG cells impairs larval
development and lipid endosomal trafficking
79
5-Discussion
81
6-References
88
3
Abstract
4
________________________________________________________________Abstract
Parasitic wasps attack a number of insect species on which they feed, either
externally or internally. This requires very effective strategies for suppressing the
immune response and a finely tuned interference with the host physiology that is coopted for the developing parasitoid progeny. The wealth of physiological host
alterations is mediated by virulence factors encoded by the wasp or, in some cases, by
polydnaviruses (PDVs), unique viral symbionts injected into the host at oviposition
along with the egg, venom and ovarian secretions. PDVs are among the most powerful
immunosuppressors in nature, targeting insect defense barriers at different levels.
During my PhD research program I have used Drosophila melanogaster as a model to
expand the functional analysis of virulence factors encoded by PDV focusing on the
molecular processes underlying the disruption of the host endocrine system. I focused
my research on a member of the ankyrin (ank) gene family, an immunosuppressant
found in bracovirus, which associates with the parasitic wasp Toxoneuron nigriceps. I
found that ankyrin disrupts ecdysone biosynthesis by impairing the vesicular traffic of
ecdysteroid precursors in the cells of the prothoracic gland and results in developmental
arrest.
5
1-Introduction
6
___________________________________________________________1-Introduction
Starting from the second half of the last century a responsible use of planet’s
natural resources to protect the environment for future generations has become a
priority.
In this context, a major issue which must be drastically reduced is the indiscriminate use
of pesticides to control food production. While the control of economically important
pest insects is still largely based on the use of chemical insecticides, the use of
biological control agents is a valid alternative. Bio-insecticides, natural molecules
deriving from bacteria, viruses, plants and animals, are environmentally safe,
biodegradable and have much higher specificity than chemical pesticides, which instead
display a wide spectrum of negative effects on all organisms, including humans.
These considerations stimulated an increase of studies aimed at the identification,
isolation, characterization and production of molecules that could be used as bioinsecticides. In particular, strong efforts have been directed towards investigations of
the control strategies used by insect’s natural enemies.
Among these enemies, parasitoids are attracting special interest because they have
developed an impressive range of sophisticated strategies of host colonization which
often eventually kill the parasitized hosts. Adult females lay their eggs in or on host
bodies, where maternal and virulence factors create favorable conditions for the
development of parasitoid progeny. Therefore, the study of physiological and molecular
mechanisms underlying these host-parasitoid associations promises to yield candidate
bioactive molecules that could be used as means to control pests attacking a wide
variety of commercial crops.
7
___________________________________________________________1-Introduction
1.1 Parasitoids
Parasitoids are entomophagus insects that parasitize other arthropods exploiting
their host for both nourishment and reproduction, and at the same time damaging it.
Their life style falls between parasitism and predation: they lay their eggs either at the
surface or into an arthropod host, generally another insect, often, but not necessarily,
during the larval or pupal stages, perform their own larval development at its expense,
and end-up killing their host as predators.
Even if various organisms use the parasitoid life style, it is mainly studied in
holometabolous insects. Although there are parasitoid insect species in six different
orders (Diptera, Coleoptera, Lepidoptera, Trichoptera, Neuroptera and Strepsiptera),
more than 80% of the described species are Hymenoptera (Quicke, 1997).
Parasitoids have developed a huge variety of strategies to colonize their hosts through
specialized mechanisms generated by long adaptive processes occurred within hostparasitoid interactions (Vinson and Scott, 1974; Vinson and Iwantsch, 1980; Godfray,
1994; Quicke, 1997).
Based on their specific colonization features, parasitoids can be classified for example
as solitary or gregarious depending on the number of eggs laid, as ectoparasitoids which
feed outside the host body, and endoparasitoids, which feed inside the host body
(Godfray, 1994); and as koinobionts and idiobionts depending on their behavior.
Idiobiont females block host development by injecting specific secretions which are
able to preserve host tissues and/or facilitate its digestion by their larvae. Koinobionts,
instead, allow host growth until the maturation of their own progeny is complete.
Koinobionts include the so called conformers, endoparasitoids that conform their own
development to host physiology, and the regulators, endoparasitoids able to alter host
physiology to create an environment suitable for successful egg development.
8
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Generally, regulators parasitize early host stages and modulate host physiology,
morphology and development, redirecting host metabolism to their own advantage.
Therefore, a close anatomic-physiological interaction is established between the host
and the parasitoid, which generally shows a significant degree of morphological
simplification combined with a high degree of specialization (Pennacchio and Strand,
2006). The latter is converted in the association of the parasitoid species with only a
given host species, or a wider but homogenous systematic group. This has led, in turn,
to the evolution of fine regulatory mechanisms allowing the parasitoid to evade host
immune defenses.
Host regulation is exerted by the action of both maternally-derived and embryonic
factors. For example, there are special polyploid cells, the teratocytes, generated by the
dissociated embryonic membrane when the egg hatches. These cells circulate freely
within the host’s haemolymph and they grow in size without undergoing cell division
(Pennacchio and Strand, 2006). These cells influence host metabolic and endocrine
balance, allowing parasitoid development.
Maternal factors of host regulation consist of venom and ovarian fluid proteins. They
are injected into the host during oviposition, and play a key role in the induction of the
major alterations observed in parasitized hosts. In certain wasp groups the ovarian fluids
also contain a symbiotic virus of the family Polydnaviridae.
9
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1.2 Polydnaviruses
Polydnaviruses (PDVs) are among the major host regulation factors used by
parasitic wasps to subdue their hosts. They function as immunosuppressant and cause a
number of developmental and reproductive alterations associated with disruption of the
host’s endocrine balance (Turnbull and Webb, 2002). These parasitic insects have a
peculiar injection device, the ovipositor, which is used to deliver the egg along with
viral particles into the host body (Figure 1) (Beckage and Gelman, 2004). Unlike most
viruses, PDVs are not transmitted by infection, indeed no virus replication occurs in
parasitized host tissues. PDVs are integrated as proviruses in the genome of parasitoid
wasps and their transmission to offspring is strictly vertical, through the germline.
Figure 1. Life cycle integration of PDV and parasitoid wasps. 1: PDV genes are integrated
into wasp chromosomes, but replicate only in female wasp calyx cells. 2: Female wasp injects
eggs and PDV into host. 3: PDV is expressed in wasp host, suppressing its immune system and
facilitating parasitoid development. 4: Parasitoid wasp larva kills its host during emergence
(Webb et al., 2006).
10
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The genome encapsidated in the PDV viral particles is made of multiple circular
dsDNA segments, which have an aggregate size ranging between 190 and 600 kb
(Webb and Strand, 2005).
PDVs can only replicate in pupal and adult stages in the epithelial cells of the calyx, a
specific region of the ovary, where they accumulate to a high density and are injected at
oviposition, along with the venom and the egg. Free viral particles infect the host tissues
and express virulence factors that alter host physiology in ways essential for offspring
survival (Strand, 2010).
The Polydnaviridae are a unique family of insect viruses with peculiar molecular
features of their genomes and with obligate association with endoparasitoid wasps
(Stoltz et al., 1984; Volkoff et al., 2002). They are composed of two genera with distinct
evolutionary origins, bracoviruses and ichnoviruses, associated with braconid and
ichneumonid wasps, respectively, (Turnbull and Webb, 2002). Bracovirus-bearing wasp
species have a common ancestor. The classical hypothesis is that bracoviruses originate
from an ancestor virus initially integrated into the genome of the ancestor wasp species
that lived 73.7 ± 10 million years ago (Whitfield, 2002). The encapsidated genomes of
all PDVs have clear eukaryotic architectural features characterized by low coding
density and many intron-containing genes, which are often members of gene families
(Espagne et al., 2004; Webb et al., 2006). Evolutionary convergence has favored the
selection of gene families shared by both ichnoviruses and bracoviruses, among which,
protein tyrosine phosphatases (PTP) and ankyrin motif proteins (ANK), widely
distributed in many PDVs and expressed to different degrees in virtually all host tissues
analyzed so far (Strand, 2012b). The wide distribution of these genes indicates that they
play a key role in successful parasitism. However, the mechanistic aspects involved
11
___________________________________________________________1-Introduction
have been only marginally characterized to date, largely in terms of immune
suppression and developmental alterations (Strand, 2012a).
1.3 The host-parasitoid association Heliothis virescens - Toxoneuron nigriceps
The host-parasitoid complex Heliothis virescens (Fabricius) (Lepidoptera,
Noctuidae) - Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) is one of the
better characterized model systems so far (Beckage, 1993).
T. nigriceps, formerly in the genus Cardiochiles (Whitfield and Dangerfield, 1997), is
an endophagous parasitoid of the tobacco budworm H. virescens larvae (Figure 2).
Figure 2. Toxoneuron nigriceps parasitizes a tobacco budworm larva, Heliothis virescens
(Pennacchio et al., 2003).
T. nigriceps females can successfully parasitize host at any larval stage from first to
early fifth instar, but parasitoid development is arrested in the first instar until the host
reaches the last larval stadium, at which point the parasitoid molts to the second instar
12
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(Pennacchio et al., 1993). Parasitized H. virescens last instar larvae fail to pupariate and,
as it happens in other host-parasitoid associations, they show the suppression of the
immune system and alterations in the endocrine balance (Pennacchio and Strand, 2006).
The modified biochemical profile of haemolymph from parasitized H. virescens leads to
an increased concentration of proteins, resulting in a better nutritional environment for
the developing parasitoid larvae (Pennacchio et al., 2003).
The larval developmental arrest of the host is due to a peak of juvenile hormone (JH)
(Li et al., 2003), associated with the suppression of 20-hydroxyecdysone (20E)
production, and gradual accumulation of polar ecdysteroids (Pennacchio et al., 2001).
These hormonal alterations are caused by maternal (calyx fluid and venom) and
embryonic-derived (teratocytes) host regulatory factors of parasitic origin. The
combined action of maternal secretions and T. nigriceps-associated bracovirus (TnBV)
inactivates the prothoracic gland in last instar host larvae, without altering the PTTH
production (Tanaka and Vinson, 1991), while the teratocytes transform the 20E into
inactive polar compounds (Pennacchio et al., 1994). It has been demonstrated that
teratocytes also produce parasitism-specific proteins (PSP) that are released into the
host haemolymph. PSP are involved in the inhibition of host immune reaction, in the
facilitation of the parasitoid larva egression by aiding on the digestion of the host
cuticle, since T. nigriceps larva lacks an elaborated mandibular apparatus, or in the
accumulation of host nutrients to support parasitoid larval development (Consoli et al.,
2007).
Furthermore, the hormonal disruption in H. virescens larvae after T. nigriceps
parasitization prevents normal replacement of the midgut epithelium, which undergoes
cell death during the fifth instar, making available additional nutritional resources for
parasitoid larval development (Tettamanti et al., 2008). Host proteins represent a rich
13
___________________________________________________________1-Introduction
source of aromatic amino acids that may be required at the end of parasitoid larval
development.
1.4 The Braconidae polydnavirus associated with T. nigriceps (TnBV)
TnBV is a typical polydnavirus, showing a segmented genome, made of circular
dsDNA molecules, which range in size from 2.5 kb to 23 kb (Varricchio et al., 1999).
PDV genes can be classified into three classes according to their different spatial and
temporal expression patterns. Class I genes are expressed in the wasp during virus
replication, class II genes are expressed in the lepidopteran host after parasitization and
class III genes are expressed in both hosts (Theilmann and Summers, 1988). Class I
genes are thought to be associated with virus replication, class II genes with disruption
of lepidopteran physiology and class III genes with unspecified functions that may be
important in both hosts (Webb, 1998). Most of identified and characterized TnBV genes
belong to class II.
The first TnBV gene isolated (TnBV1) is small in size and contains a single intron.
TnBV1 cDNA encodes for a putative protein of 124 amino acids with a molecular mass
around 15 kDa. This protein shows no striking sequence similarity with other known
proteins and it contains one potential N-linked glycosylation site, one potential
phosphorylation site for cAMP and cGMP-dependent protein kinases, and two protein
kinase C phosphorylation sites. The TnBV1 gene produces a transcript of about 0.5 kb
starting from 12 h after parasitoid oviposition, which abundance increases rapidly and
peaks between 24 h and 48 h after parasitization (Varricchio et al., 1999). The TnBV1
transcript is present, exclusively in parasitized host tissue, in both fat body and
haemocytes, showing a much higher level of expression in haemocytes. The transient
expression of this gene in different cell types, especially in haemocytes, suggests that it
14
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may have a role in immune suppression. Also, detection of TnBV1 mRNA in the host’s
prothoracic gland indicated a possible influence on endocrine and developmental
disruption (Malva et al., 2004).
The second TnBV gene (TnBV2) characterized (Falabella et al., 2003) contains two
small exons and a small intron encoding for a putative protein composed of 153 amino
acids, with a calculated molecular mass of 18 kDa. The gene produces a transcript of
0.6 kb in parasitized hosts, as early as 6 h after parasitoid oviposition, and its abundance
increases rapidly and reaches the maximum level between 24 h and 48 h after
parasitization. The same 0.6 kb transcript is present, 48 h after parasitoid oviposition, in
various host tissues, such as fat body, haemocytes, prothoracic gland and the
head/thorax region (Falabella et al., 2003). A conserved retroviral type aspartic protease
domain (Rawlings and Barrett, 1995), from amino acid 42 to amino acid 119, with the
characteristic Asp–Thr–Gly active site, is located in a conserved region in the TnBV2
gene product (Malva et al., 2004).
The major gene family identified in the TnBV genome codes for protein tyrosine
phosphatases (PTPs) with 13 members expressed in host different tissues. The large
number of genes in the bracovirus PTP families, the complex profile of their expression
and their key role, along with tyrosine kinases, in the regulation of signal transduction
pathways (Andersen et al., 2001), suggest that these proteins may induce the alteration
of several physiological traits of parasitized hosts (Falabella et al., 2006).
Two cDNAs have been identified in this gene family: the first (TnBVPTP5) codes for a
putative protein composed of 294 amino acids and the second (TnBVPTP7) codes for a
putative protein composed of 293 amino acids, both with a calculated molecular mass of
34.7 kDa (Falabella et al., 2006). At 24 h post-parasitsm, TnBVPTP5 and TnBVPTP7
are expressed in haemocytes and in fat body from the thorax region of the host larvae
15
___________________________________________________________1-Introduction
and only the TnBVPTP7 is also observed in prothoracic gland. This suggests that PTPs
expression may be tissue- and/or substrate-specific (Falabella et al., 2006). The
presence of PTPs in the haemocytes of parasitized host larvae (Provost et al., 2004)
suggests that these genes reinforce and maintain the haemocyte inactivation
mechanisms triggered soon after parasitoid oviposition by the combined action of the
venom and TnBV expression (Ferrarese et al., 2005).
Another gene family found in the bracovirus associated with T. nigriceps includes
members which code IκB-like proteins (ANK). These putative proteins, consisting
mostly of ankyrin repeats, display significant sequence similarity (approximately 50%)
with members of the IκB protein family, which act as inhibitors of NF-κB signaling
pathways in insects and vertebrates (Silverman and Maniatis, 2001), but lack the
regulatory domains controlling their signal-induced degradation (Falabella et al., 2007).
In Drosophila, the IκB protein Cactus regulates multiple cellular responses activated by
the nuclear import of a set of NF-κB/Rel proteins, which control embryonic
dorsoventral patterning (Bergmann et al., 1996) and antimicrobial response (De
Gregorio et al., 2001; Hoffmann, 2003).
In parasitized H. virescens larvae the ANK proteins, along with ovarian proteins, venom
components and other PDV products, bind irreversibly to NF-κB/Rel immunoreactive
proteins which function is repressed by cytoplasmic sequestration. These larvae are
unable to encapsulate foreign invaders because of the PDV-induced disruption of NFκB signaling that possibly affects both the humoral and cellular immune responses
(Falabella et al., 2007).
Three open reading frames were found by genome sequencing in TnBV that were
denoted TnBVank1–3. TnBVank1 and TnBVank3 were isolated from a library prepared
with mRNAs extracted from haemocytes of parasitized H. virescens larvae, but not
16
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TnBVank2. This result indicates that TnBVank2 may correspond to a pseudogene or may
be expressed and functional in a different host (Falabella et al., 2007).
1.5 TnBVank1
TnBVank1 is located on a 4.7 kb genome segment and codes for a putative
protein composed of 155 amino acids with a calculated molecular mass of 17.1 kDa.
The TnBVANK1 protein predicted from its isolated cDNA is made up of an ankyrin
domain comprising four repeats, which show similarity with repeats 3–6 of Cactus and
IκBα (Figure 3) (Falabella et al., 2007). Interestingly, TnBVANK1, like the other viral
ANK proteins, does not contain the N-terminal IKK target motif that mediates the
signal-induced degradation of Cactus or the C-terminal PEST domain, present in the
Cactus/IκB proteins, which is involved in rapid protein turnover (Rogers et al., 1986).
Figure 3. Schematic representation of the protein encoded by TnBVank1 gene compared
with the ankyrin repeats of human (Hum) IκBα and Drosophila (D.) Cactus. The GenBank
accession numbers are indicated below the name of the proteins. The numbers under each
protein scheme indicate the amino acid positions delimiting the different ankyrin repeats.
HumIκBα regulatory regions: SRD, signal-receiving domain mediating phosphorylation and
ubiquitination; PEST, region responsible for rapid protein turnover; NES, leucine-rich nuclearexport sequences, NLS, nuclear-localization signal. Modified from (Falabella et al., 2007).
17
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In the haemocytes of H. virescens larvae after parasitization by T. nigriceps, TnBVank1
was transcribed very early (3 h post-parasitization) and the level of its expression
declined clearly by 48 h post-parasitization (Falabella et al., 2007).
1.6 Drosophila melanogaster as a model system
From the pioneering studies of T. H. Morgan and members of his laboratory in
the early 1900s, Drosophila melanogaster has early become the most characterized
model organism for genetic studies due to the relative simplicity of its genome, the
shortness of its life cycle and the abundance of progeny.
With the development of molecular biology techniques and genome sequencing, the
fruit fly has served as an excellent model system for studying the mechanisms
regulating essential biological processes and has had a major role in unraveling the
molecular mechanisms at the basis of almost all metazoan development and physiology.
The availability of genome sequences, the ease of genetic manipulation, and the large
collection of available mutants make Drosophila an attractive system that has enabled a
better understanding of different diseases at the molecular level (Niwa and Niwa, 2011).
More recently, studies on hormonal homeostasis and metabolism have also been
performed in this excellent genetic model organism. Similar to vertebrates, insects
require cholesterol as a precursor for steroid hormones and as a structural component of
cell membranes (Niwa and Niwa, 2011). The regulation of ecdysteroidogenesis has
been studied continuously for the past several decades and recent discoveries using
Drosophila molecular genetics have advanced our knowledge. For example, studies
with both invertebrates and vertebrates revealed an important conserved property in
steroidogenesis, the involvement of cytochrome P450 enzymes (CYPs) (Beckstead and
Thummel, 2006; Rewitz et al., 2006b). Thus, studying steroidogenesis in insects, and in
18
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particular in the model organism Drosophila melanogaster, can not only help gain
insights into the biosynthesis of steroid hormone but may also provide valuable
information about the regulation of insect development.
1.7 Overview of Drosophila development
The Drosophila life cycle is summarized in Figure 4. At 25° C the development
from egg to adult takes approximately 10 days and includes four distinct developmental
phases: egg, larva, pupa and adult. Embryonic development lasts for about 24 h, after
which a feeding larva hatches from the egg. The larvae undergo three successive stages,
referred to as larval instars: L1, L2 and, L3, which last for about 24 h, 24 h, and 48 h,
respectively. At the end of the third instar, larvae cease to feed and enter a wandering
stage in search of a suitable site for pupariation. The pupal phase holds over the next
four days and on the fifth day flies emerge from their pupal cases (Mulinari, 2008).
Figure 4. The life cycle of Drosophila melanogaster. At 25°C the complete life cycle lasts
approximately for 10 days. Larvae molt through three larval instars before metamorphosing into
their adult form (Hartwell et al., 2011).
19
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The larva is characterized by two cellular types: larval cells, that are polyploid, and
imaginal cells that are diploid. Imaginal cells segregate precociously from the
surrounding larval cells, forming small cell groups at 9-10 h after egg deposition and are
organized in two fundamental groups, imaginal discs and abdominal histoblasts.
Imaginal discs begin an intense proliferative activity from the second larval instar until
pupariation, while abdominal histoblasts proliferate later, during the pupal stage. At this
moment the majority of larval cells are eliminated and substituted by imaginal cells that
originate the integument and the adult appendages. Imaginal discs originate the
structures of the head, thorax external appendages, genitalia and adult muscles. The
histoblats originate the abdomen structures at the exception of the 8th segment that
derives from the genital imaginal disc (Fristrom and Fristrom, 1993). The primary
mechanism by which the larva grows is molting. At each molt the entire cuticle of the
insect, including its many specialized structures, as well as the mouth armature and the
spiracles, is shed and rebuilt again. During each molt, therefore, many reconstruction
processes occur leading to the formation of structures characteristic of the ensuing
instar. The growth of the internal organs proceeds gradually and seems to be largely
independent of the molting process, which mainly affects the body wall. Organs such as
Malpighian tubes, muscles, fat body, intestine and ring gland grow by an increase in cell
size; the number of cells in the organ remains constant. The imaginal discs, on the other
hand, grow chiefly by cell multiplication and the size of the individual cells remains
about the same (Deepa Parvathi et al., 2009).
A series of developmental steps by means of which the insect passes from the larval into
the adult organism is called “metamorphosis”. The most drastic changes in this
transformation process take place during the pupal stage. The larva everts its anterior
spiracles and becomes motionless. Metamorphosis involves the destruction of certain
20
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larval tissues and organs (histolysis) and the organization of adult structures from
imaginal discs (Demerec and Kaufman, 1996). The duration and extent of these
transformation processes vary greatly for the different organs involved. Larval organs
which are completely histolyzed during metamorphosis are the salivary glands, the fat
bodies, the prothoracic gland, the intestine and apparently the muscles. The extremities,
eyes, mouthparts, antennae, and genital apparatus differentiate from their appropriate
imaginal discs, which were already present in the larval stage and which undergo
histogenesis during pupal development (Milislav, 1950). When metamorphosis is
complete, the adult flies emerge from the pupal case.
1.8 The role of the steroid hormone ecdysone in Drosophila melanogaster
The transformation from larva to adult is one of the most fascinating processes
of insect biology and is characterized by different developmental phenomena, including
cellular proliferation, tissue remodeling, cell migration and programmed cell death.
Cells undergo one or more of these processes in response to hormone secretion. In
particular, in Drosophila most of developmental processes are governed primarily by
ecdysone (E) and juvenile hormone (JH) whose balance likely affects the nature of
developmental transitions. In particular, the JH has a classic “status quo” action in
preventing the program-switching action of ecdysone during larval molts (Riddiford et
al., 2010).
The molting process is initiated by the brain, where two pairs of neurosecretory cells
release prothoracicotropic hormone (PTTH) in response to neural, hormonal or
environmental signals. PTTH itself is synthesized as a pre-prohormone, processed
intracellularly to its final size (109 amino acids) and released into the hemolymph as a
21
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homodimeric molecule containing intra- and intermolecular disulfide bonds. This
neuropeptide stimulates the prothoracic gland to produce ecdysone.
The PTTH transduction cascade has been well elucidated in lepidopteran model insects
where the interaction of PTTH with its receptor (Torso in Drosophila) at the cell
membrane surface of prothoracic gland cells involves phospholipase C (PLC),
phosphatidylinositol-4,5-biphosphate
(PIP2),
inositol
triphosphate
(IP3)
and
diacylglycerol (DAG). PIP2 stimulates protein kinase C (PKC) while IP3 elicits the
release of calcium from the endoplasmic reticulum into the cytosol where it can act to
open both the store operative and L-type voltage gated channels so that even more Ca2+
can enter the cell. The increase of calcium is followed by an intracellular cAMP level
rising mediated by Ca2+-calmodulin dependent adenylyl cyclase activity and the protein
kinase A (PKA), important cAMP-dependent kinase, is rapidly activated (Figure 5).
22
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Figure 5. The PTTH signal transduction cascade in prothoracic gland cells. Solid lines
indicate demonstrated or highly likely interactions; dashed lines indicate hypothetical
relationships. PTTH, prothoracicotropic hormone; PLCβ, phospholipase C β; PIP2,
phosphatidylinositol-4,5-biphosphate; DAG, diacylglycerol; IP3, inositol triphosphate; GTP,
guanosine triphosphate; cAMP, cyclic adenosine monophosphate; CaM, calmodulin; AdCyc,
adenylyl cyclase; ER, endoplasmic reticulum; PKA, protein kinase A; PI3K, phosphoinositide
3hydroxy-dependent kinase; TOR, target of rapamycin; MEKK, MEK kinase; MEK, MAP/ERK
kinase; ERK, extracellular signal-regulated kinase; S6, ribosomal protein S6; p70S6K, 70 kDa
S6 kinase; MNK 1, MAP kinase-interacting kinase; Ras, a small GTP binding protein; Raf, a
serine-threonine kinase; elF-4E, eukaryotic translation initiation factor 4E (Huang et al., 2008).
23
___________________________________________________________1-Introduction
Of great importance is the phosphorylation in five sites of ribosomal protein S6 which
can result in the selective translation of specific mRNAs required for ecdysone
biogenesis.
PTTH also activates the MAPK pathway resulting in a rapid increase of ERK
phosphorylation, indicating that ecdysteroidogenesis in the prothoracic gland requires
the presence of a small basal population of di-phosphorylated (active) ERK molecules
(Gilbert, 2004).
During larval stages, ecdysone is produced and released from the prothoracic gland,
while in the adult after histolisys of the PG, itis produced in the fat body and in the
ovary.
Once released into the hemolymph, ecdysone is modified in peripheral tissues to
become the active molting hormone 20-hydroxyecdysone (20E).
Each molt is initiated by one pulse of 20E (Figure 6). For a larval molt, the first pulse
produces a small rise in the 20E concentration in the larval hemolymph and elicits a
change in cellular commitment. A second, large pulse of 20E initiates the differentiation
events associated with molting. The 20E produced by these pulses commits and
stimulates the epidermal cells to synthesize enzymes that digest and recycle the
components of the cuticle.
24
___________________________________________________________1-Introduction
Figure 6. The life cycle of Drosophila melanogaster at 25°C in the context of changing
ecdysteroid hormone titer. The fruit fly develops through three larval stages before it reaches
puparium formation. The larval stages are separated by molts, which are controlled by pulses of
ecdysone. Other major developmental events, such as hatching and the transition from larva to
pupa, are also controlled by this hormone.
Juvenile hormone is secreted by the corpora allata, one of the two parts of the ring
gland. The secretory cells of the corpora allata are active during larval molts and
inactive during the metamorphic molt. As long as JH is present, the 20E-stimulated
molts result in a new larval instar. In the last larval instar, however, the medial nerve
from the brain to the corpora allata inhibits the gland from producing JH, and there is a
simultaneous increase in the body’s ability to degrade existing JH (Safranek and
Williams, 1989). Both these mechanisms cause JH levels to drop below a critical
threshold value. This triggers the release of PTTH from the brain (Nijhout and
25
___________________________________________________________1-Introduction
Williams, 1974) while the resulting 20E, in the absence of high levels of JH, commits
cells to pupal development.
After the second ecdysone pulse, new pupa-specific gene products are synthesized
(Riddiford, 1982) and the subsequent molt shifts the organism from larva to pupa. It
appears, then, that the first ecdysone pulse during the last larval instar triggers the
processes that inactivate the larva-specific genes and prepare the pupa-specific genes to
be transcribed. The second ecdysone pulse transcribes the pupa-specific genes and
initiates the molt (Nijhout, 1994). At the imaginal molt, when ecdysone acts in the
absence of juvenile hormone, the imaginal discs differentiate, and the molt gives rise to
the adult. Larval tissues such as the gut, salivary glands, and larval-specific muscles
undergo programmed cell death and subsequent histolysis. The imaginal discs undergo
physical restructuring and differentiation to form rudimentary adult appendages such as
wings, legs, eyes and antennae (Jiang et al., 1997).
Hence, ecdysone tightly coordinates the array of physiological changes that characterize
each stage of the life cycle. Interestingly, while all tissues are exposed to the hormone,
different tissue types have unique responses to the signal. This is ensured by the
different spatial and temporal expression profile and unique biochemical properties of
its nuclear receptors and by the ability of these receptors to interact with many
cofactors.
The ecdysone signal is transduced to target genes in the genome via the ecdysone
receptor complex. This complex is made up of a heterodimer of the Ultraspiracle
protein (USP) (Oro et al., 1990; Shea et al., 1990; Henrich et al., 1994) and the
Ecdysone Receptor (EcR) proteins (Koelle et al., 1991; Koelle, 1992; Yao et al., 1992;
Talbot et al., 1993; Thomas et al., 1993; Yao et al., 1993). The EcR/USP complex binds
ecdysone and affects transcription of ecdysone target genes. This molecular interaction
26
___________________________________________________________1-Introduction
is the means by which ecdysone regulates the genes responsible for the plethora of
physiological changes that are characteristic of the developmental progression through
the life cycle. These early genes encode transcription factors that coordinate the
induction of large sets of secondary-response late genes, leading to the appropriate stage
and tissue-specific biological responses (Figure 7) (Ashburner et al., 1974).
Figure 7. The Ashburner model. Ecdysone binding to its receptor initially activates the
expression of genes in the early puff regions, but represses the expression of those in the late
puffs. As the proteins encoded by the early puff genes become abundant, they repress their own
promoters while activating the expression of late puff genes (Tata, 2002).
Ecdysone regulates a wide range of developmental and physiological responses in
Drosophila, including reproduction, oogenesis, embryogenesis, post-embryonic
development other than metamorphosis. Ecdysone also triggers neuronal remodeling in
the central nervous system (Schubiger et al., 1998).
Moreover, it has been demonstrated that ecdysone controls larval growth rate and final
adult size and this function is mediated by an antagonistic interaction with insulin
signaling (Colombani et al., 2005).
27
___________________________________________________________1-Introduction
1.9 The prothoracic gland: the site of ecdysteroidogenesis
The Drosophila larval ring gland lies above the brain hemispheres with its dorsal
portion tilted anteriorly. Its lateral extremities encircle the aorta like a ring, hence the
name. The ring gland consists mainly of the prothoracic gland which comprises the
anterior and lateral portions of the two limbs of the ring gland, the corpora allata, source
of juvenile hormone, which is located in the anterior medial area, and the corpora
cardiaca, a neurohemal organ, which occupies the posterior ends of the two limbs
(Figure 8) (Dai and Gilbert, 1991).
Figure 8. Cartoon of the Drosophila ring gland. The ring gland is composed of the
prothoracic gland (PG; yellow), the corpora allata (CA; orange) and the corpora cardiaca (CC;
red).
It is the prothoracic gland portion of the ring gland that is responsible for the synthesis
of ecdysteroids that, in turn, elicit the sequence of events termed molting. This gland
usually begins to degenerate during pharate adult life (Wigglesworth, 1955; Herman
and Gilbert, 1966) since the molt from pupa to adult is normally the last molt in the life
of an insect. At the early wandering third instar, ultrastructural observation reveal some
distinct features of an active prothoracic gland cell including abundant tubular smooth
28
___________________________________________________________1-Introduction
endoplasmic reticulum, numerous and various shaped mitochondria and large and deep
intercellular channels (Dai and Gilbert, 1991).
The Drosophila ring gland does not persist in the adult fly; in fact it undergoes drastic
changes during larva-pupa and pupa-adult transitions. Particularly, the prothoracic gland
degeneration is a gradual process initiated after puparium formation. Individual
prothoracic gland cell of the same gland also show differential sensitivity during the
process, so that the ring gland retains a reduced, but significant, ability to synthesize
ecdysteroids during the period of gland demise. However, by 30-40 h after puparium
formation, the majority of gland cells are undergoing cell death, characterized by the
presence of numerous giant autophagic vacuoles in the cytoplasm and fragmentation of
the cytoplasm (Dai and Gilbert, 1991).
1.10 Ecdysone biosynthesis
Among all gene categories required for ecdysteroidogenesis, the enzymes
needed for converting cholesterol to 20E are best known, including a group of seven
genes named “Halloween”: spook (spo), spookier (spok) shroud (sro), phantom (phm),
disembodied (dib), shadow (sad) and shade (shd) (Jurgens et al., 1984; NussleinVolhard et al., 1984; Wieschaus et al., 1984; Ono et al., 2006; Niwa et al., 2010). These
genes have likely been highly conserved since the presence of these genes has been
confirmed in other insect species (Niwa et al., 2004; Warren et al., 2004; Warren et al.,
2006) and encode cytochrome P450 enzymes that are believed to act sequentially in the
biosynthesis of ecdysone (Figure 9).
29
___________________________________________________________1-Introduction
Figure 9. Scheme of the biosynthesis of 20E in Drosophila. The 7, 8-dehydrogenase may be
encoded by the Rieske non-heme oxygenase gene neverland. spook and spookier encode for a
P450 enzyme that may operate in the Black Box along with the product encoded by shroud.
Modified from (Huang et al., 2008).
30
___________________________________________________________1-Introduction
In Drosophila, synthesis of ecdysone during larval stages takes place primarily within
the prothoracic gland cells, while the conversion of ecdysone to 20E occurs in other
tissues including the midgut and fat body (Gilbert, 2004). In the adult female, 20E is
required for proper oogenesis and follicle cells appear to be an additional major site of
ecdysone production. Consistent with these tissues being the major sources of ecdysone
and 20E, the Drosophila phm, dib and sad genes, are all expressed in the prothoracic
gland cells of the ring gland beginning midway through embryogenesis and show
periodic expression within this tissue during larval stages (Chavez et al., 2000; Warren
et al., 2002; Niwa et al., 2004; Warren et al., 2004; Warren et al., 2006). During the
third instar, expression of these genes correlates well with the hemolymph ecdysteroid
titer (Parvy et al., 2005; Warren et al., 2006). In adult female flies, all three genes show
pronounced expression in the follicle cells of the ovary beginning at approximately
stage 8 of oogenesis. In contrast, the Drosophila shd gene is not expressed in the
prothoracic gland cells, but instead is found in peripheral target tissues such as the
epidermis, midgut, Malpighian tubules and fat body, where Shd mediates conversion of
ecdysone into the active hormone 20E (Petryk et al., 2003; Rewitz et al., 2006a). As
with the other three enzymes, however, shd is also expressed in the follicle cells of the
ovary, consistent with a 20E requirement for normal oogenesis (Buszczak et al., 1999;
Carney and Bender, 2000; Terashima et al., 2005).
The Halloween gene spo is expressed in both the follicle cells of the ovary and in the
early embryo prior to the differentiation of the ring gland but is not expressed at
detectable levels in the prothoracic gland cells of the ring gland throughout the
remainder of embryonic and larval development. This surprising difference in the
temporal expression of spo compared to that of other Halloween genes suggested that
Drosophila must have a gene that codes for an enzyme that should substitute for the Spo
31
___________________________________________________________1-Introduction
protein during larval stages. This gene turns out to be spookier (spok). Spok is about
57% identical to the Drosophila Spo protein and displays the highest degree of identity
between all Drosophila P450 proteins. As such, Spo and Spok are paralogs and so likely
code for P450 enzymes that catalyze the same reaction. spok is expressed in the
prothoracic gland cells of the ring gland (with phm, dib and sad) from embryonic stage
16 through to the end of larval development and continuing through to the end of pupaladult development, but not in the adult ovary, exactly complements the expression of
spo. Just like phm, dib and sad, during larval development, spok expression peaks in the
ring gland at the end of the each instar, falls at the beginning of the next, to rise again
with the rising ecdysteroid titer (Ono et al., 2006).
Unlike vertebrate steroidogenesis, where all the metabolites between cholesterol and the
various active steroid hormones have been isolated and identified, no intermediate
between the initial hydrogenation product of cholesterol (7-dehydrocholesterol) and the
first recognizable ecdysteroid-like product (ketodiol) has been observed in Drosophila.
These reactions are still a “black box” as defined by Dennis Horn (Gilbert, 2011). spo
and spok, which encode CYP307A1 and CYP307A2 respectively, may operate in the
black box converting 7-dehydrocholesterol to Δ4-diketol and then Shroud reduces Δ4diketol to ketodiol. The following biosynthetic reactions are fully understood. In the
prothoracic gland cells there are Phm (CYP306A1; 25-hydroxylase) which converts
ketodiol to ketotriol, Dib (CYP302A1; 22-hydroxylase) that converts ketotriol to 2deoxyecdysone and Sad (CYP315A1; 2-hydroxylase) which converts 2-deoxyecdysone
to ecdysone. The final reaction occurs in the target tissues where Shd (CYP314A1; 20hydroxylase) transforms ecdysone in 20E (Figure 9).
Consistent with their sub-cellular localization and enzymatic function (Kappler et al.,
1988), both Phm and Spo/Spok are in the endoplasmic reticulum according to their
32
___________________________________________________________1-Introduction
characteristic N-terminal string of hydrophobic residues, while Dib, Sad and Shd
localize at the mitochondria.
Recently, another gene has been characterized whose expression is also up-regulated in
the prothoracic gland during the last instar of Drosophila development: neverland (nvd)
which encodes a Rieske non-heme iron oxygenase. Like most of the Halloween genes, it
is expressed specifically in both the embryonic and larval prothoracic gland and in the
nurse cells of the developing adult ovary. Here too, its expression varies in step with
both the haemolymph ecdysteroid titer and prothoracic gland activity (Yoshiyama et al.,
2006). As Rieske-oxygenases have been shown to catalyze a multitude of reactions,
including desaturation reactions, it is possible that Nvd catalyzes the long-sought
cholesterol to 7-dehydrocholesterol reaction in Drosophila (Yanagawa et al., 2011).
Over the last years, many other genes were identified and characterized, like without
children (woc), molting defective (mld) and ecdysoneless (ecd) (Gaziova et al., 2004;
Neubueser et al., 2005). Since these genes do not encode enzymatic products, it has
been proposed that they may have a role in regulating ecdysone biosynthesis and in the
transfer of the biosynthetic intermediate between cellular organelles.
1.11 Cholesterol trafficking in steroidogenic cells
Since cholesterol is the substrate for steroidogenesis, its uptake, transport and
trafficking must be crucial for ecdysone biosynthesis.
There are two general sources of cellular cholesterol: dietary uptake and de novo
synthesis. Cells maintain proper cholesterol homeostasis through a negative feedback:
once dietary derived cholesterol level is high, de novo synthesis is inhibited and vice
versa. However, in order to regulate de novo synthesis, dietary derived cholesterol must
33
___________________________________________________________1-Introduction
go through multiple steps (intestinal uptake, intercellular transport and intracellular
trafficking) before reaching the endoplasmic reticulum (Niwa and Niwa, 2011).
It has been known that insects are not able to synthesize sterols from simple precursors
and thus depend solely on dietary sterol uptake for ecdysteroid biosynthesis and other
cellular needs (Clark and Block, 1959). Insects take up phytosterols (such as
campesterol and β-sitosterol) through the intestinal absorption. Then the intestinal
derived sterol is transported peripherally via haemolymph. As in the case of mammalian
cells, insect cells take up exogenous cholesterol through the classic receptor-mediated
low density lipoprotein (LDL) endocytic pathway. LDL binds to its receptor, LDLR,
which is then internalized into the endosome. LDL is then released, while LDLR
recycles back to the plasma membrane. Once in the endosomal compartment, esterified
cholesterol in the LDL is hydrolyzed by lipase to free cholesterol which then leaves the
endosomal compartment to move to other membrane compartments including the
endoplasmic reticulum, plasma membrane and mitochondria for various cellular usages
(Figure 10) (Rodenburg and Van der Horst, 2005).
34
___________________________________________________________1-Introduction
Figure 10. Regulating sterol availability for ecdysteroidogenesis. Dietary sterols are
transported in the midgut as lipoprotein particles to the ring gland cells. The lipoprotein
particles move into the cell through receptor-mediated endocytosis, then to the endosomal
system where sterols are released and transported subsequently to Golgi, ER and mitochondria
for ecdysone biosynthesis. EE: early endosome; LE: late endosome; ER: endoplasmic
reticulum; N: Nucleus; MT: mitochondria. Modified from (Huang et al., 2008).
35
2- Research Aims
36
_________________________________________________________2-Research Aims
PDVs are unique viral obligate symbionts of parasitic wasps injected into the
host at oviposition along with the egg, venom and ovarian secretions. PDVs infect the
host tissues and express virulence factors that alter host physiology in ways essential to
offspring survival. These virulence factors suppress the immune response in parasitized
lepidopteran larvae and impair development and/or reproduction. To date, most studies
have focused on the molecular mechanisms underpinning immunosuppression, whereas
how viral genes disrupt the endocrine balance remains largely uninvestigated.
During my PhD research program I have analyzed a member (TnBVank1) of the ankyrin
gene family of the bracovirus associated with Toxoneuron nigriceps, a larval parasitoid
of the noctuid moth Heliothis virescens. The aim of my studies was to gain insight into
the molecular mechanisms through which TnBVANK1 acts as a virulence factor
disrupting host endocrine system.
To this purpose, I analyzed the effects of TnBVank1 during Drosophila development.
Drosophila provides an excellent model system to investigate fundamental topics linked
to endocrine system because of the relative simplicity of its signaling pathways.
Moreover, the well-established genetic and genomic tools define the fruit fly as an ideal
model system for studying the molecular mechanisms of essential biological processes.
I focused my studies on directing the expression of TnBVank1 in the prothoracic gland
by using the Gal4/UAS binary system. In particular, I analyzed the involvement of this
viral ankyrin protein in the cholesterol endosomal trafficking, the first step of ecdysone
hormone synthesis.
37
3-Materials and Methods
38
__________________________________________________3-Materials and Methods
3.1 Fly food
The Drosophila melanogaster strains used were grown on a corn meal based
food supplemented with glucose, yeast, agar and water. The food is prepared by melting
10 gr of agar and 50 gr of glucose in 1600 ml water. Then 150 gr of corn meal are added
and food is cooked for 15 minutes mixing well. Afterward 50 gr of yeast are added and
food is cooked for 10 minutes more, again on medium and mixing well. While food is
cooking, 4 gr of Nipagine, an antimicotic, are dissolved in 16 ml of EtOH 98% and
added. The food is left to dry for at least 2 hours.
3.2 Fly strains
The following stocks are used:

y1,w67c23 as a wild-type stock;

UASp-TnBVank1/UASp-TnBVank1;UASp-TnBVank1/UASp-TnBVank1;+/+
generated in our lab (Duchi et al., 2010). In the text the transgene will be
referred as UASp-TnBVank1;

1734: w*;+/+;P{GawB}h1J3 (Brand and Perrimon, 1993), (Bloomington
Drosophila Stock Center). In the text the transgene will be referred as hairyGal4;

1878: w*;P{GawB}T80/CyO;+/+ (Wilder and Perrimon, 1995), (Bloomington
Drosophila Stock Center). In the table will be referred as T80-Gal4;

5073: w*;+/+;P{UAS-p35.H}BH2 (Zhou et al., 1997), (Bloomington Drosophila
Stock Center). In the text will be referred as UAS-p35;

5535: w*;P{GAL4-ey.H}4-8/CyO;+/+ (Carrera et al., 1998), (Bloomington
Drosophila Stock Center). In the table will be referred as Eyeless-Gal4;
39
__________________________________________________3-Materials and Methods

6357: y1w1118;+/+;P{Lsp2-GAL4.H}3 (Roignant et al., 2003), (Bloomington
Drosophila Stock Center). In the text and in the table will be referred as Lsp2Gal4;

6479:
y1w*;P{GawB}sca109-68/CyO;+/+
(Manning
and
Doe,
1999),
(Bloomington Drosophila Stock Center). In the table will be referred as
Scabrous-Gal4;

6990: w1118;+/+;P{GawB}C855a (Hrdlicka et al., 2002), (Bloomington
Drosophila Stock Center). In the table will be referred as c885a-Gal4;

7019: w*;P{w+mC=tubP-GAL80ts}20;TM2/TM6B,Tb1 (Davis et al., 2003),
(Bloomington Drosophila Stock Center). In the text the transgene will be
referred as tub-Gal80ts;

7374: y1w*;P{UASp-GFPS65C-αTub84B}14-6-II;+/+ (Grieder et al., 2000),
(Bloomington Drosophila Stock Center). In the text will be referred as UASp-αtubulin-GFP;

8700: w*;+/+;P{He-GAL4.Z}85,P{UAS-GFP.nls}8 (Zettervall et al., 2004),
(Bloomington Drosophila Stock Center). In the table will be referred as
Hemese-Gal4;

8760: w*;+/+;P{GAL4-elav.L}3 (Luo et al., 1994), (Bloomington Drosophila
Stock Center). In the table will be referred as Elav-Gal4;

30140:
w1118;P{Hml-GAL4.Δ}2,P{UAS-2xEGFP}AH2;+/+
(Sinenko
and
Mathey-Prevot, 2004), (Bloomington Drosophila Stock Center). In the table will
be referred as Hemolectin-Gal4;

32040:
P{Appl-GAL4.G1a}1, y1w*;+/+;+/+
(Torroja
et
al.,
1999),
(Bloomington Drosophila Stock Center). In the table will be referred as ApplGal4;
40
__________________________________________________3-Materials and Methods

32119: w*;+/+;P{GawB}337Y (Manseau et al., 1997), (Bloomington Drosophila
Stock Center). In the table will be referred as 337Y-Gal4;

v32047: w1118;P{GD7853}v32047;+/+ (Dietzl et al., 2007), (Vienna Drosophila
RNAi Center). In the text will be referred as UAS-ALIX-RNAi;

yw;+/+;phantom-Gal4,UAS-mCD8GFP/TM6B, kindly provided by C. Mirth
(Mirth et al., 2005). In the text will be referred as phantom-Gal4;

w*;+/+;P0206-Gal4,UAS-mCD8GFP, a gift from C. Mirth (Mirth et al., 2005).
In the text will be referred as P0206-Gal4;

yw;august21-Gal4/CyO;+/+ was generated in our lab from the stock
yw;august21-Gal4/CyO;phantom-Gal4/phantom-Gal4 kindly provided by M.
Jindra. In the text will be referred as august21-Gal4.
3.3 Genetic crosses
Females UASp-TnBVank1 were crossed to males of the different Gal4 lines. As
control, females y1,w67c23 were crossed to males of the same Gal4 lines.
For microtubules analysis, females UASp-TnBVank1 were crossed to males UASp-αtubulin-GFP;phantom-Gal4.
To coexpress p35 and TnBVANK1 in PG cells females UASp-TnBVank1;UASpTnBVank1;UAS-p35 were crossed to males phantom-Gal4,UAS-mCD8GFP/TM6B.
All the previous crosses are performed at 25°C.
For Gal80ts experiment, females UASp-TnBVank1 were crossed to males tubGal80ts;phantom-Gal4,UAS-mCD8GFP/TM6B at 21°C and then the resulting larvae
were shifted to 31°C at 96 h, 72 h and 48 h after egg deposition (AED).
41
__________________________________________________3-Materials and Methods
3.4 Larval length measurements
Five UASp-TnBVank1/+;UASp-TnBVank1/+;hairy-Gal4/+ larvae at different
days AED and five control larvae were ice-anesthetized and photographed using a Nikon
Eclipse 90i microscope. Images were taken at 4X magnification and the larval length
was measured with NIS-Elements Advanced Research 3.10 software.
3.5 20-hydroxyecdysone (20E) titration
This analysis was performed by Professor Sheng Li’s group of the Institute of
Plant Physiology & Ecology (Shanghai).
Five larvae at different developmental stages were collected and washed with PBS
buffer and immediately frozen by liquid nitrogen. Samples were added 200 μl of
methanol, homogenized and transferred into 1.5 ml plastic tubes. After 10 minutes
centrifugation (12,000 rpm at 4°C) the supernatant was collected into a new tube, the
precipitate was re-extracted with 200 μl of methanol and the supernatant was added to
the previous one. After 30 minutes on ice, the samples were centrifuged following the
same conditions. Samples were dried to remove methanol and then dissolved in the
borate buffer. The standard curve was generated according to the standard process of the
RIA protocol (Warren et al., 2006) and then the 20E titer in samples was calculated.
3.6 Rescue experiments
UASp-TnBVank1/+;UASp-TnBVank1/+;phantom-Gal4,UAS-mCD8GFP/+
larvae and controls were collected at 106 hours AED and placed in three groups of ten
individuals at 25°C in new tubes supplemented with 20E (Sigma) dissolved in ethanol at
1 mg/ml. Control larvae were fed only with ethanol.
42
__________________________________________________3-Materials and Methods
3.7 Prothoracic gland and cellular size measurements
For measurements of PG area and its cellular size, confocal images of 50 PGs
taken at 40X magnification were quantified with ImageJ software.
3.8 Immunofluorescence Microscopy
Larvae were dissected at room temperature in phosphate buffer saline (1xPBS)
pH 7.5 and fixed in 4% formaldehyde in 1xPBS pH 7.5 for 20 minutes at room
temperature. After three washes 5 minutes each in 1xPBS pH 7.5, larvae were
permeabilized in 1xPBT (1xPBS pH 7.5 + 0.3% Triton X-100) for 1 hour, washed three
times 5 minutes each in 1xPBT and 10 minutes in 1xPBT + 2% BSA solution. After
that, the larvae were incubated, overnight at 4°C, with primary antibodies diluted in
1xPBT + 2% BSA. Next day larvae were washed three times 10 minutes each in 1xPBT,
10 minutes in 1xPBT + 1% BSA solution and incubated at room temperature on a
rotating wheel with secondary antibodies diluted in 1xPBT + 1%BSA. After three
washes 5 minutes in 1xPBT, the ring glands were dissected and mounted on microscopy
slides in Fluoromount G (Electron Microscopy Sciences), an anti-quenching slide
mounting
medium.
Subsequently
samples
were
analyzed
by
conventional
epifluorescence with a Nikon Eclipse 90i microscope or with TCS SL Leica confocal
system. Images were processed using Adobe Photoshop CS4.
TRITC-Phalloidin staining was carried out, after incubation with secondary antibodies,
by washing larvae three times 5 minutes each with 1xPBS pH 7.5 and then by incubating
larvae for 20 minutes at room temperature with TRITC-Phalloidin (40 μg/ml in 1xPBS
pH 7.5; Sigma). After three washes 5 minutes each in 1xPBS pH 7.5, the ring glands
were dissected and mounted as indicated above.
43
__________________________________________________3-Materials and Methods
For Propidium Iodide nuclear counterstaining, the larvae were treated with RNase A
(400 g/ml in 1xPBT; Sigma) overnight at 4°C. After three washes 10 minutes each in
1xPBT, the larvae were labeled for 2 hours with Propidium Iodide (10 g/ml in 1xPBT;
Molecular Probes) then the ring glands were dissected and mounted in Fluoromount G.
3.9 Antibodies
The following primary antibodies were used in this study:
 polyclonal rabbit anti-Disembodied, Dib, kindly provided by M. O’Connor
(Parvy
et
al.,
2005),
was
raised
against
the
peptide
KTLLINKPDAPVLIDLRLRREC of the Drosophila Disembodied protein and
was used at 1:200;
 polyclonal rabbit anti-Without children, Woc, a gift from M. Gatti (Raffa et al.,
2005), recognizes a specific domain of the Drosophila Without children protein
(aa 230-626) and was used at 1:500 dilution;
 polyclonal rabbit anti-TnBVANK1 (Duchi et al., 2010) is directed against two
TnBVANK1
peptides,
(LLGERNELGNNFFHE)
one
located
and
the
other
at
the
at
the
N-terminal
domain
C-terminal
domain
(NDKKMMEILKKNGAK), was used at 1:200 dilution;
 polyclonal rabbit anti-Cleaved Caspase-3 (9661, Cell Signaling Technology,
(Fernandes-Alnemri et al., 1994)) detects endogenous levels of a large fragment
(17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175
and was used at 1:50 dilution;
 sheep anti-Digoxigenin-fluorescein (1207741, Roche) is directed against the
whole Digoxigenin protein and conjugated with 5(6)-carboxy-fluorescein-Nhydroxy-succinimide ester (FLUOS) and was used at 1:100 dilution;
44
__________________________________________________3-Materials and Methods
 monoclonal mouse P1H4 anti-Dynein heavy chain (McGrail and Hays, 1997)
recognizes a region of the cytoplasmic dynein heavy chain protein (aa 128-422)
and was used at 1:200 dilution;
 monoclonal mouse anti-Rab5 (610281, BD Biosciences) recognizes human Rab5
(aa 1-215) and was used at 1:25 dilution;
 polyclonal rabbit anti-Rab7, kindly provided by A. Nakamura (Tanaka and
Nakamura, 2008), recognizes a region of Rab7 protein (aa 184-200) and was
used at 1:2000 dilution;
 polyclonal rabbit anti-Rab11, kindly provided by A. Nakamura (Tanaka and
Nakamura, 2008), recognizes a region of Rab11 protein (aa 177-191) and was
used at 1:5000 dilution;
 polyclonal guinea pig anti-Hrs, a gift from T. Lloyd (Lloyd et al., 2002),
recognizes the amino-terminal half (aa1-376) of the Hrs protein and was used at
1:1000 dilution;
 monoclonal mouse anti-ALIX, kindly provided by T. Aigaki (Tsuda et al.,
2006), was raised against the full length of ALIX protein and was used at 1:100
dilution.
Secondary antibodies:

rabbit antibodies were detected with Cy3-conjugated sheep anti-rabbit used
1:1000 (Sigma), Cy3-conjugated goat anti-rabbit used 1:1000 (Jackson),
DyLight 649-conjugated goat anti-rabbit used 1:500 (Jackson) and BODIPYconjugated goat anti-rabbit used 1:2000 (Sigma);
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__________________________________________________3-Materials and Methods

mouse antibodies were detected with Cy3-conjugated goat anti-mouse used
1:1000 (Jackson) and DyLight 649-conjugated goat anti-mouse used 1:500
(Jackson);

guinea pig antibodies were detected with DyLight 649-conjugated goat antiguinea pig used 1:500 (Jackson).
3.10 Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling
(TUNEL) Analysis
Five days AED larvae were dissected at room temperature in 1xPBS pH 7.5,
fixed in 4% formaldehyde in 1xPBS pH 7.5 for 20 minutes at room temperature. After
three washes 5 minutes each in 1xPBS pH 7.5, larvae were dissected and permeabilized
for 1 hour with a solution of 1xPBS pH 7.5 + 0.3% Triton X-100 (1xPBT) at room
temperature. After three washes 5 minutes each with 1xPBT, larvae were incubated in
250 l of TUNEL solution (5X Reaction Buffer, 25mM CaCl2, 1mM d-UTPdigoxigenin, 25U/l TdT) for 90 minutes at 37°C in dark condition. Afterward, larvae
were accurately washed several times with 1xPBT and then for 10 minutes in 1xPBT +
2% BSA. Next, larvae were incubated with Cy3-conjugated-digoxigenin antibody for 2
hour at room temperature on a rotating wheel. After incubation, larvae were washed
many times with 1xPBT, the ring glands were dissected and mounted in Fluoromount
G.
3.11 Filipin and Oil Red O stainings
Larvae were dissected at room temperature in 1xPBS pH 7.5, fixed in 4%
formaldehyde in 1xPBS pH 7.5 for 20 minutes and washed three times in 1xPBS pH 7.5
for 5 minutes each. Samples were stained with 50 μg/ml of Filipin (Sigma) for 1 hour or
46
__________________________________________________3-Materials and Methods
incubated in an Oil Red O (Sigma) solution at 0.06% for 30 minutes. After incubation
larvae were washed twice with 1xPBS pH 7.5 for 5 minutes each, the ring glands were
dissected and mounted in Fluoromount-G. Samples were analyzed by conventional
epifluorescence with a Nikon Eclipse 90i microscope or with a Nikon Eclipse 90i
confocal microscope. Images were processed using Adobe Photoshop CS4.
3.12 Colocalization analysis
Thresholds of confocal images were set in Adobe Photoshop CS4 to exclude
background staining. 509 Hrs positive vesicles were analyzed per TnBVANK1 and Hrs
staining. 443 TnBVANK1 positive vesicles were analyzed per TnBVANK1 and ALIX
staining. 118 Hrs positive vesicles were analyzed per ALIX and Hrs staining.
Images were processed with the CDA plugin of ImageJ to obtain Pearson’s coefficient
(from +1=complete correlation, to -1=anti-correlation with 0=no correlation) (Zinchuk
and Zinchuk, 2008).
3.13 Statistical analysis
Statistical comparison of mean values was performed by unpaired t-test, using
GraphPad Prism 4 software.
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4-Results
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4.1 The expression of TnBVank1 arrests development during larval stage three
In order to explore the function of the TnBVank1 gene, I induced its expression
during Drosophila development through the Gal4/UAS binary system (Brand and
Perrimon, 1993). This system is based on two genetic elements kept in different lines:
the yeast transcriptional activator Gal4 downstream of a given promoter (driver) and the
Gal4-dependent UAS cis-regulatory sites upstream of the target gene (responder).
When responder and driver lines are brought together by crossing, the resulting progeny
express the responder in the transcriptional pattern of the driver.
I used a transgenic Drosophila stock carrying two copies of the TnBVank1 gene under
the control of UASp sequences, which allow the expression either in the somatic cells or
in the female germline (Figure 11) (Duchi et al., 2010).
Figure 11. The TnBVank1 expression is directed using the Gal4/UAS system. Crossing flies
of the driver line with the stock carrying the TnBVank1 gene under the control of UASp
sequences, the resultant progeny expresses the TnBVank1 transgene in specific tissues.
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The expression of the TnBVank1 transgene was induced using different Gal4 drivers. I
expressed this transgene during embryonic and larval development using the hairy-Gal4
driver (h-Gal4) (Brand and Perrimon, 1993). This line expresses the UAS-linked genes
during the embryonic development and in different larval tissues as brain, ring gland,
salivary gland, imaginal discs and midgut. All the h-Gal4>TnBVank1 larvae completed
the embryonic and larval development but, interestingly, failed to pupariate and died
after an extended third instar, which lasted up to three weeks (Figure 12A). At four days
after egg deposition (AED), when the last larval stage starts, these larvae did not
significantly differ in size from the control yw;h-Gal4 ones (n=5; t=0.8557; NS) (Figure
12B). Interestingly, while control larvae regularly pupariated on day six AED (Figure
12A), the larvae expressing TnBVank1 continued to feed and significantly increased in
size during their prolonged larval life, reaching the maximal length at eighteen days
(Figure 12A, C; n=5; t=6.765; p<0.0001).
The observed developmental arrest suggested me that TnBVANK1 could have
reasonably affected the hormonal endocrine system. Particularly, since h-Gal4 is
expressed in various larval tissues among which the ring gland, the expression of
TnBVank1 gene in this organ could cause the block during development.
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Figure 12. Larvae expressing TnBVank1 fail to pupariate and continue to grow. (A) Light
micrographs of yw;h-Gal4 larva and pupa (control) and h-Gal4>TnBVank1 larvae at different
days AED. (B) The larval length of five larvae of different genotypes is measured at 96 h AED.
Graph represents mean ± standard deviation (SD). There is no significant (NS) length difference
between h-Gal4>TnBVank1 (blue; 2680±83 μm) and yw;h-Gal4 larvae (red; 2580±82 μm). As
additional controls (patterned yellow), the larval length of yw and h-Gal4 and UASp-TnBVank1
stocks is measured. (C) Five h-Gal4>TnBVank1 larvae are monitored during their extended
third instar and their larval length is analyzed at different days AED. Values are the mean ± SD
of three independent experiments. The mean values of h-Gal4>TnBVank1 larval length at four
and eighteen days AED are shown above the bars.
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4.2 TnBVank1 expression in the prothoracic gland cells blocks the larva-to-pupa
transition
As described in detail in paragraph 1.9, the Drosophila ring gland is the major
site of production and release of developmental hormones. It is composed of the
prothoracic gland (PG), which synthesizes the ecdysone; the corpora allata (CA) that
produce the juvenile hormone, and the corpora cardiaca (CC), which play a key role in
the regulation of metabolic homeostasis (Figure 8) (Dai and Gilbert, 1991). To verify if
the expression of TnBVank1 in the ring gland were able to reproduce the effect observed
when the transgene was expressed using the h-Gal4, I targeted its expression using
different ring gland-specific Gal4 drivers (Figure 13). When the TnBVank1 gene was
expressed in both CA and PG cells using the P0206-Gal4 driver, the larval development
arrested during the third instar showing the same phenotype obtained with h-Gal4.
Conversely, when the august21-Gal4 (aug21-Gal4) driver specifically targeted the
expression of TnBVank1 in the CA, no effects on development were observed and
regular progeny were obtained. Finally, I specifically induced the expression of
TnBVank1 gene in the PG using the phantom-Gal4 (phm-Gal4) driver, which is strongly
expressed in this gland. All the larvae failed to pupariate and presented an extended life
as shown using the P0206-Gal4 driver. Therefore, the block of larval-pupal transition is
due to the activity of TnBVANK1 in the prothoracic gland cells.
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Figure 13. Effects of TnBVank1 expression in the ring gland. The expression of the
TnBVank1 gene is driven in the different ring gland compartments, highlighted in green, by
three Gal4 drivers. P0206-Gal4, expressed in PG and CA, causes the developmental arrest at
the last larval stage; aug21-Gal4 (CA) does not induce any developmental defects; phm-Gal4
(PG) blocks the transition from larval to pupal stage.
Recently, it has been demonstrated that in insects tissue damage is frequently associated
with a systemic injury response, resulting in a delay of development as prolonged larval
or pupal stages (Hackney et al., 2012). To verify that this was not the case with the
expression of TnBVank1 in specific tissues, I expressed the transgene in several other
tissues using different Gal4 drivers. Monitoring the timing of development and the adult
phenotype, no effects were observed in all cases analyzed (Table 1).
Collectively, these data suggest that the expression of TnBVANK1 has the potential to
interfere with the steroid biosynthesis, as further indicated by the targeted expression of
this viral ANK protein in the PG, which is characterized by developmental arrest of last
instar larvae.
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Table 1. Effects of TnBVank1 expression in specific tissues on developmental timing. All
the Gal4 drivers used are identified by the Bloomington stock center number. For each cross,
the percentage (%) of normal adults is calculated by dividing the number of normal adults by
the total number of animals of the same genotype.
4.3 phm-Gal4>TnBVank1 larvae contain low levels of 20-hydroxyecdysone
The developmental arrest induced by TnBVANK1 during the third instar could
be due to an insufficient level of 20E to trigger the puparium formation. In order to
verify this hypothesis the whole body 20E titer in phm-Gal4>TnBVank1 and in control
larvae (Figure 14A) was measured by a collaboration with Professor Sheng Li’s group
of the Institute of Plant Physiology & Ecology (Shanghai) who performed this analysis.
At 25°C wild type third instar larvae enter the wandering stage at 110 h AED and then,
after the surge of a 20E peak, become white pre-pupae at 120 h AED (Warren et al.,
2006). The 20E level measured in phm-Gal4>TnBVank1 larvae is extremely reduced
and significantly lower than that measured both in UASp-TnBVank1 larvae (n=5;
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t=10.12; p<0.0001) and in phm-Gal4/TM6B larvae (n=5; t=8.196; p<0.0001) at 120 h
AED. Moreover, it keeps low during their abnormal extended larval life.
To further demonstrate that the block of transition from larval to pupal stages showed
by the phm-Gal4>TnBVank1 larvae was actually due to a low level of 20E, I carried out
an ecdysteroid-feeding rescue experiment. At 25°C, third instar phm-Gal4>TnBVank1
larvae were fed with yeast paste containing 20E dissolved in ethanol at 106 h AED, just
before the onset of the ecdysteroid peak occurring in the wild-type. As expected, at 120
h AED, 70% of yw;phm-Gal4 control larvae started to pupariate and within the
following 20 h all of them reached the pupal stage (n=30). Pupariation of phmGal4>TnBVank1 larvae fed with 20E followed an almost identical pattern, with 100%
pupariation (n=30) attained only 1-day later, but failed to progress to the pharate stage
(Figure 14B). Instead, phm-Gal4>TnBVank1 larvae treated only with yeast and ethanol
persisted as third instar individuals (n=30). This result confirmed that the developmental
arrest of phm-Gal4>TnBVank1 larvae was due to a reduced level of 20E. However, the
rescued pupae failed to develop into adult flies. This may be due to the fact that the
large peak of 20E required to trigger metamorphosis was not generated by phmGal4>TnBVank1 pupae and cannot obviously be supplied with food at this
developmental stage.
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Figure 14. In phm-Gal4>TnBVank1 there are low levels of 20E. (A) Total 20E titer in UASpTnBVank1 (red bars), phm-Gal4/TM6B (cyan bars) and phm-Gal4>TnBVank1 (black bars)
larvae at different developmental stages. In the control stocks UASp-TnBVank1 and phmGal4/TM6B, the 20E peak which induces the pupariation is present at the white prepupal stage
(120 h AED). Instead, this peak is absent in phm-Gal4>TnBVank1 larvae and the total 20E titer
remains low during the extended larval life. Error bars represent SD; ***=p < 0.0001 versus
controls (UASp-TnBVank1 and phm-Gal4/TM6B). The mean values of total 20E at 120 h AED
of different genotype larvae are shown above the bars. (B) Feeding phm-Gal4>TnBVank1
larvae with medium supplemented with 20E induces the pupariation (orange), while phmGal4>TnBVank1 larvae fed with medium containing ethanol (EtOH) do not reach the pupal
stage (green). The yw;phm-Gal4 larvae serve as background control (blue).
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Recently, it has been reported that in the late third instar the genes involved in ecdysone
biosynthesis in PG are up-regulated to support high ecdysone production (Moeller et al.,
2013). To evaluate if the up-regulation of these gene were present in phmGal4>TnBVank1 PG, I analyzed the expression and localization of Disembodied (Dib),
the 22-hydroxylase which converts ketotriol to 2-deoxyecdysone. I dissected the PGs of
yw;phm-Gal4 and phm-Gal4>TnBVank1 larvae at 120 h AED, during the ecdysone
peak for pupariation, and I performed an immunostaining with anti-Dib antibody (Parvy
et al., 2005). Interestingly, Dib was strongly reduced in phm-Gal4>TnBVank1 cells
compared to controls (Figure 15A, B). Another gene essential for the ecdysone
biosynthesis is neverland which encodes for the cholesterol 7,8-dehydrogenase. Its
transcription is controlled by the transcription factor Without children (Woc) (Warren et
al., 2001). In PGs dissected from yw;phm-Gal4 and phm-Gal4>TnBVank1 larvae at 120
h AED I analyzed the expression of Woc by immunostaining (Raffa et al., 2005). This
transcription factor was largely localized in the nucleus in control PG cells (Figure
15C), whereas it was present at reduced levels and was also localized in the cytoplasm
in phm-Gal4>TnBVank1 PG cells (Figure 15D). Thus, in phm-Gal4>TnBVank1 PG at
120 h AED Dib and Woc were not up-regulated and these data consolidate the low
levels of 20E in phm-Gal4>TnBVank1 larvae.
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Figure 15. The expression of Dib and Woc is reduced in PG of phm-Gal4>TnBVank1
larvae. Immunostaining with anti-Dib in yw;phm-Gal4 (A) and phm-Gal4>TnBVank1 (B) PG
reveals that the expression of Dib is strongly reduced in phm-Gal4>TnBVank1 larvae. (C) The
transcription factor Woc is normally localized in the nucleus, as shown in yw;phm-Gal4 PG. (D)
Instead, in phm-Gal4>TnBVank1 PG Woc is present also in cytoplasm and its expression level
is reduced, while in CA it is strictly nuclear. All confocal images are at the same magnification
and the reference scale bar is shown in A.
4.4 The expression of TnBVank1 affects the PG morphology
In order to investigate the expression and the distribution of TnBVANK1 in PG
cells I performed an immunostaining using a polyclonal antibody raised against two
synthetic peptides of TnBVANK1 (Duchi et al., 2010). To visualize the PG I used the
phm-Gal4,UAS-mCD8GFP driver line which expresses the membrane-bound GFP only
in the PG cells. As shown in Figure 16, the TnBVANK1 protein was strongly detected
only in the cytoplasm of PG cells, confined to stroke-shaped particles.
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Figure 16. The distribution of TnBVANK1 in the PG cells. The immunolocalization of
TnBVANK1 (cyan) in PG cells (marked with mCD8GFP), shows its presence in stroke-shaped
particles distributed only in the cytoplasm. In B and C nuclei are stained with Propidium Iodide
(red). Confocal images in B and C are at the same magnification and the reference scale bar is
shown in B.
Moreover, analyzing these glands I observed that PGs from control larvae (Figure 17A)
were significantly larger (n=50; t=50.41; p<0.0001) (Figure 17C) than phmGal4>TnBVank1 PGs (Figure 17B). Measurements of the PG cell area did not show a
significant reduction in phm-Gal4>TnBVank1 cells compared to control cells (Figure
17D). Therefore, the observed size difference of PG can be attributed to a reduction of
cell number that may be caused by the TnBVANK1-induced apoptosis in some PG
cells.
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Figure 17. phm-Gal4>TnBVank1 PG are smaller than yw;phm-Gal4 PG. Confocal images
of five days AED PG, marked with mCD8GFP of yw;phm-Gal4 (A) and phm-Gal4>TnBVank1
(B) larvae. (C) By measuring the PG size, phm-Gal4>TnBVank1 larvae have significantly
smaller PGs than control. The graph represents the mean ± SD; 50 PGs were analyzed;
***=p<0.0001. (D) No differences in PG cell area were observed (50 PGs analyzed, NS: nonsignificant).
In order to assay if apoptosis occurs in phm-Gal4>TnBVank1 PG, I performed an
immunostaining on PGs dissected at 120 h AED using Cleaved Caspase-3 antibody
(Florentin and Arama, 2012) and TUNEL labeling assay (Gavrieli et al., 1992).
The Caspase-3 activity (Figure 18B, B’; n=60) and the TUNEL positive staining (Figure
18D, D’; n=60) found in some cells of the phm-Gal4>TnBVank1 PGs, and not detected
60
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in control PGs (Figure 18A, C), suggested that the occurrence of cell death during
development can partly account for this difference. Thereby, the size reduction of phmGal4>TnBVank1 could be related to the developmental arrest induced by TnBVANK1.
Figure 18. Apoptosis in phm-Gal4>TnBVank1 PG. Confocal images of immunostaining with
anti-Cleaved Caspase-3 (A-B’, red) and TUNEL (C-D’, red) in PG cells marked with
mCD8GFP. In the control yw;phm-Gal4 no caspase (A) or TUNEL (C) signals are detected,
while in phm-Gal4>TnBVank1 PG few cells undergo apoptosis (B, B’, D, D’). PGs in panels A,
B, C, D are at the same magnification and the scale bar is shown in A. Boxed regions are
magnified in B’ and D’ and the reference scale bar is shown in B’.
However, the possibility that TnBVANK1 can also disrupt the PG steroidogenic activity
cannot be ruled out. Therefore, to assess the relative contribution of these two effects,
which could be not mutually exclusive, I expressed the transgene TnBVank1 in PG cells
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at different time points during larval life, using a temperature-sensitive form of the Gal4
repressor Gal80, Gal80ts (McGuire et al., 2003). Gal80 represses activation of Gal4 by
binding specifically to its activation domain, and its temperature-sensitive mutant
Gal80ts is active at 21°C but does not repress Gal4 at 31°C (Figure 19). This system
allowed me to regulate the phm-Gal4 activity throughout development.
Figure 19. Schematic representation of the UAS/Gal4 and Gal80ts system used to regulate
the expression of TnBVank1 in the PG. The temperature-sensitive Gal80 protein (Gal80ts),
expressed ubiquitously from the tubulin promoter, represses the transcriptional activity of Gal4
at 21°C and thus prevents the expression of the UAS-TnBVank1 transgene in the PG cells. At
31°C, Gal80ts becomes inactive and allows Gal4 to drive the expression of the UASTnBVank1 transgene in the PG.
UASp-TnBVank1;UASp-TnBVank1/tub-Gal80ts;phm-Gal4/+
larvae
and
yw;tub-
Gal80ts/+;phm-Gal4/+ control larvae were initially raised at 21°C, and then shifted to
the restrictive temperature (31°C) at specific time points (96 h, 72 h and 48 h AED) to
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promote Gal4 activity. The temperature shift did not affect the proper development of
the control larvae, which pupariated normally. Conversely, the larvae expressing
TnBVank1 failed to pupariate, increased their size and survived for an extended period.
For each time point I also analyzed the PG size at 120 h AED (Figure 20). When the
TnBVank1 expression was triggered at 96 h or 72 h, the PG size was not significantly
different from the control (respectively n=10; t=0.07636; NS and n=10; t=1.336; NS).
Instead, the earlier induction of the transgene expression, at 48 h AED, strongly affected
the PG size, which appeared significantly reduced (n=10; t=11.68; p<0.0001).
Figure 20. The reduction of PG size is due to an early induction of TnBVank1 expression.
yw;tub-Gal80ts/+;phm-Gal4/+ larvae (control) and UASp-TnBVank1;UASp-TnBVank1/tubGal80ts;phm-Gal4/+ (Gal80ts-TnBVank1) larvae are raised at 21°C (cyan) for different time
intervals, then shifted at 31°C (red) and their PG dissected at 120 h AED. PG size from Gal80tsTnBVank1 larvae incubated at 21°C until 96 h AED or until 72 h AED shows no significant
(NS) differences from that of control larvae. PG size is strongly reduced in Gal80ts-TnBVank1
larvae incubated at 21°C until 48 h AED compared to PG from control larvae (***=p<0.0001).
Graph represents mean ± SD; 10 PGs were analyzed for each experiment.
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In addition, I examined whether ectopic expression of the anti-apoptotic protein p35
(Hay et al., 1994) should rescue the phenotype produced by the expression of TnBVank1
in the PG. Co-expression of UAS-p35 and UASp-TnBVank1 in the same PG cells
through the phm-Gal4 driver did not rescue the developmental arrest phenotype (n=58).
Collectively, these data indicate that the developmental arrest induced by TnBVANK1
does not depend on the reduced PG size caused by apoptosis, but on its capacity to
disrupt the PG steroidogenic function when expressed before the production of the 20E
peak.
4.5 The expression of TnBVank1 in the PG impairs the cytoskeletal network
The phm-Gal4>TnBVank1 PG cells revealed a cytoplasmic rather than the
expected membrane distribution of mCD8GFP, as showed in Figure 17B (Lee and Luo,
1999). The observed mislocalization of mCD8GFP and the altered morphology of phmGal4>TnBVank1 PG prompted me to analyze the cytoskeletal network in these cells. I
dissected PGs from phm-Gal4>TnBVank1 and yw;phm-Gal4 larvae at 120 h AED and I
evaluated the organization of the cytoskeleton in PG cells by analyzing F-actin and αtubulin distribution. The organization of F-actin was evaluated by phalloidin staining
(Sigma) (Figure 21A, A’, D, D’). The cortical actin did not appear regularly distributed
in phm-Gal4>TnBVank1 PG cells, in which thick masses of actin filaments were
detected (Figure 21D, D’). The microtubule network was investigated by analyzing the
distribution of an α-tubulinGFP fusion protein, which was co-expressed with TnBVank1
in the PG. In the control, the PG cells expressed only α-tubulinGFP protein (Figure
21B, B’). The microtubule cytoskeleton of the phm-Gal4>TnBVank1 PG cells appeared
strongly affected, as shown by the formation of thick bundles of microtubules (Figure
21E, E’). The dynamic function of the microtubule network was then analyzed by
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assessing the distribution of the minus-end-directed microtubule motor Dynein, using an
anti-Dynein heavy chain antibody (McGrail and Hays, 1997). Compared to the yw;phmGal4 cells (Figure 21C, C’), the phm-Gal4>TnBVank1 PG cells displayed a reduced
cortical distribution of Dynein, along with some large Dynein dots (Figure 21F, F’).
These data indicate that the whole cytoskeletal network is markedly altered in the PG
cells expressing TnBVANK1.
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Figure 21. phm-Gal4>TnBVank1 PG cells show an altered cytoskeleton. Phalloidin staining
in control (A, A’) and in phm-Gal4>TnBVank1 (D, D’) PG cells. F-actin shows an altered
distribution, characterized by thick masses of filaments in phm-Gal4>TnBVank1 PG cells. (BE’) α-tubulinGFP fusion protein was expressed in yw;phm-Gal4 and phm-Gal4>TnBVank1 PG
to investigate the microtubule network. Compared to control (B, B’), in phm-Gal4>TnBVank1
the microtubule cytoskeleton is strongly affected and forms bundles (E, E’). (C-F’)
Immunostaining with anti-Dynein heavy chain shows that, compared to control (C, C’), in phmGal4>TnBVank1 PG cells the cortical localization of this protein is reduced and characterized
by an evident dotted distribution (F, F’). Confocal images in panels A, B, C, D, E, F are at the
same magnification and the scale bar is shown in A. Boxed regions are magnified in A’, B’, C’,
D’, E’, F’ and the reference scale bar is shown in A’.
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Moreover, I analyzed the cytoskeletal structure in other tissues where the transgene
TnBVank1 was expressed. Using the driver line lsp2-Gal4, which directs the expression
of UAS-linked genes in the fat bodies, I evaluated the F-actin distribution. No
differences in phalloidin staining were observed between yw;lsp2-Gal4;UASmCD8GFP control and lsp2-Gal4>TnBVank1 fat bodies (Figure 22C, D). As shown in
Figure 22A, B the cell membrane architecture appeared regular in yw;lsp2-Gal4;UASmCD8GFP and lsp2-Gal4>TnBVank1 fat bodies.
Figure 22. Expression of TnBVank1 in fat bodies does not affect cell morphology. Confocal
images of Phalloidin staining in fat bodies from the control yw;lsp2-Gal4;UAS-mCD8GFP (A,
C) and from fat bodies expressing TnBVank1 lsp2-Gal4,UAS-mCD8GFP/TnBVank1 (B, D). Fat
bodies are at the same magnification in all panels and the scale bar is indicated in A.
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The expression of TnBVank1 in different tissues, using a wide range of tissue-specific
Gal4 drivers, did not alter the developmental timing and the adult formation, as
discussed in paragraph 4.2 (Table 1). Hence these results suggest that the cytoskeletal
structure is not affected in all tissues.
4.6 TnBVANK1 expression causes increased accumulation of lipids in PG cells
The cytoskeleton and its associated motor proteins play an important role in
protein sorting and endocytic pathways (Huotari and Helenius, 2011). Therefore the
observed negative impact of TnBVANK1 on PG cells could reduce the level of
ecdysteroid biosynthesis by disrupting the uptake, transport and trafficking of sterols,
essential steps for ecdysteroid biosynthesis (Huang et al., 2008). To evaluate if the
cytoskeletal alterations detected in PG cells may impair the endocytic pathway, I
analyzed lipid vesicular internalization and trafficking in the phm-Gal4>TnBVank1 PG
cells with a staining procedure using Oil Red O (Sigma). This fat-soluble diazol dye,
with a maximum absorption at 518 nm, stains neutral lipids and cholesteryl esters but
not biological membranes (Annika et al., 2013). Conversely to controls (Figure 23A),
phm-Gal4>TnBVank1 PG cells showed an increased accumulation of lipid droplets
(Figure 23C, C’) in all PGs analyzed (n=60). These droplets most likely include sterol
precursors required for ecdysteroid production. To better characterize this observation, I
used Filipin (Sigma) staining which specifically reveals non-esterified sterols (Friend
and Bearer, 1981). I observed that, compared to controls (Figure 23B), all phmGal4>TnBVank1 PGs analyzed (n=60) displayed a marked cholesterol accumulation in
discrete vesicular drops (Figure 23D, D’). These data suggest that TnBVANK1 does not
affect lipid uptake, but the endocytic pathway is impaired.
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Figure 23. phm-Gal4>TnBVank1 PG cells show lipids accumulation. (A) In control yw;phmGal4 there are few lipid droplets stained with Oil Red O, while in phm-Gal4>TnBVank1 PG
cells several lipid droplets are detected (C, C’). (D, D’) In phm-Gal4>TnBVank1 there is also a
sterol accumulation, shown by Filipin staining, which is absent in control PG (B). Panels A, B,
C, D are at the same magnification and the reference scale bar is shown in A. Boxed regions are
magnified in C’, D’ and the reference scale bar is shown in C’.
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4.7 The organization of the cholesterol trafficking pathway in PG cells
As discussed in detail in paragraph 1.11, cholesterol, which cannot be
synthesized by insects (Gilbert and Warren, 2005), enters in the steroidogenic cells
through a receptor-mediated low-density lipoprotein (LDL) endocytic pathway
(Rodenburg and Van der Horst, 2005), which targets cholesterol to the endosomes.
Transport to the lysosome is characterized by the maturation of the vacuolar regions of
early endosomes into late endosomes, which are able to fuse directly with the lysosome
(Figure 24).
The three major compartments of the endocytic pathway are characterized by specific
Rab GTPase proteins that can be used as tags for the different endosomes (Zerial and
McBride, 2001). Early endosomes are enriched in Rab5; Rab11 marks the recycling
endosomes and late endosomes are associated with Rab7. During the early-to-late
endosome transition, multivesicular endosomes form on early endosomal membranes
and mediate transport to late endosomes. They have thus been referred to as endosomal
carrier vesicles (ECVs) or multivesicular bodies (MVBs) according to their function or
appearance, respectively (Dikic, 2006). The Hepatocyte growth factor-regulated
tyrosine substrate (Hrs) can be used as a tag for MVBs. In fact this protein regulates
inward budding of endosome membrane and MVBs/late endosome formation (Lloyd et
al., 2002).
In late endosomes and lysosomes, acid lipase hydrolyses cholesteryl esters and the
resulting free cholesterol partition into neighboring membranes. Cholesterol is then
transformed into 7-dehydrocholesterol in endoplasmic reticulum and transported to
other
subcellular
compartments
through
further
metabolic
steps
of
the
ecdysteroidogenic pathway (Gilbert and Warren, 2005).
70
_______________________________________________________________4-Results
Figure 24. Involvement of cholesterol trafficking in insect ecdysteroidogenesis. Cholesterol
enters PG cells through LDL endocytic pathway and is then internalized by the endosomes.
Early Endosomes are marked by Rab5, Multivesicular bodies by Hrs, Recycling Endosomes by
Rab11 and Late Endosomes by Rab7. The esterified cholesterol is hydrolyzed by lipase to free
cholesterol which then leaves the endosomal compartment to move to other membrane
compartments including the endoplasmic reticulum and mitochondria. The enzymes are referred
to by their names as encoded from Halloween genes in Drosophila: Phm, phantom; Dib,
disembodied; Sad, shadow.
71
_______________________________________________________________4-Results
4.8 The endocytic pathway is altered in PG cells expressing TnBVank1
In order to investigate the endocytic pathway in PG cells I analyzed the
distribution of the endosomes. I dissected 60 PGs from yw;phm-Gal4 and phmGal4>TnBVank1 larvae at 120 h AED and I used antibodies directed against the Rab
proteins to identify the endosomes (Tanaka and Nakamura, 2008).
Immunostaining with anti-Rab5 antibody (Biosciences) showed that the distribution of
early endosomes in TnBVank1 larvae (Figure 25B) appeared to be comparable to
control PG cells (Figure 25A). Similarly, no differences in recycling endosomes, stained
by anti-Rab11 antibody (Tanaka and Nakamura, 2008), were observed in yw;phm-Gal4
(Figure 25C) and phm-Gal4>TnBVank1 (Figure 25D) PGs. Differently, few endosomal
vesicles were detected by anti-Rab7 antibody (Tanaka and Nakamura, 2008) in the
presence of TnBVANK1 (Figure 25F) compared to control (Figure 25E). This suggests
that the expression of TnBVank1 in PG cells alters the early-to-late endosome transition.
72
_______________________________________________________________4-Results
Figure 25. TnBVANK1 disrupts the endocytic pathway in PG cells. Confocal images of five
days AED PG stained for Rab5 (A, B), Rab11 (C, D) and Rab7 (E, F) in yw;phm-Gal4 (left
column) and phm-Gal4>TnBVank1 (right column) larvae. The distribution of endosomes
marked with Rab5 (A, B) and Rab11 (C, D) is not affected by TnBVank1 expression, while a
strong reduction in number was observed for late endosomes marked with Rab7 (E, F). All
panels are at the same magnification and reference scale bar is shown in A.
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_______________________________________________________________4-Results
4.9 TnBVANK1 is localized in multivesicular bodies
Since the expression of TnBVank1 in PG cells showed an altered maturation of
the early endosomes into late endosomes, I analyzed the distribution of multivesicular
bodies carrying the Hepatocyte growth factor-regulated tyrosine substrate (Hrs).
Immunostaining with anti-Hrs antibody (Lloyd et al., 2002) in yw;phm-Gal4 PGs
showed a wide cytoplasmic distribution of round shape vesicles containing this protein
(Figure 26A, A’). Interestingly, in the phm-Gal4>TnBVank1 PG cells quite a few Hrs
marked vesicles exhibited a stroke-shaped form (Figure 26B, B’), similar to the form
observed for the TnBVANK1 signal (Figure 26C, C’). I performed an immunostaining
with anti-TnBVANK1 and anti-Hrs antibodies in phm-Gal4>TnBVank1 PGs and I
found that most of the immunodetection signals of TnBVANK1 colocalized with the
Hrs-marked vesicles (Figure 26D, D’). To quantify the level of colocalization in these
PG cells, I calculated the Pearson’s correlation coefficient using the CDA plugin of
ImageJ. This coefficient, being one of standard measures in pattern recognition, was
first employed to estimate colocalization and is used for describing the correlation of the
intensity distributions between channels (Zinchuk and Zinchuk, 2008). The
TnBVANK1-Hrs Pearson’s coefficient was 0.96 ± 0.06. Since the Pearson’s coefficient
values range from -1.0 (complete separation of two structures) to +1.0 (complete
colocalization of two signals) (Zinchuk and Zinchuk, 2008), the colocalization of
TnBVANK1 and Hrs marked MVBs appears complete. In contrast, the Hrs-marked
vesicles showing a normal round shape did not colocalize with TnBVANK1. This
finding suggests an interaction of TnBVANK1 with endosome associated proteins,
which may partly account for the observed alterations of the endocytic trafficking
routes.
74
_______________________________________________________________4-Results
Figure 26. TnBVANK1 protein colocalizes with Hrs-positive vesicles. Confocal images of
PG from yw;phm-Gal4 (A, A’) and phm-Gal4>TnBVank1 (B-D’) larvae stained for Hrs (cyan)
and TnBVANK1 (red). A number of vesicles marked by Hrs in phm-Gal4>TnBVank1 cells (B,
B’) have a shape different from that present in controls (A, A’). These modified vesicles show a
strong colocalization with TnBVANK1 signal (C-D’), demonstrating that TnBVANK1 protein
is associated with Hrs-marked vesicles. PGs in panels A, B, C, D are at the same magnification
and the reference scale bar is shown in A. Boxed regions are magnified in A’, B’, C’, D’ and
their reference scale bar is shown in A’.
75
_______________________________________________________________4-Results
4.10 The role of ALIX in the endosomal trafficking
MVBs formation is controlled by a set of proteins, the endosomal sorting
complex required for transport, ESCRT-0 to III, which sequentially associates with the
cytosolic surface of endosomes (Williams and Urbe, 2007). A partner of the ESCRT
proteins is ALIX (formerly Apoptosis-Linked gene 2-Interacting protein X), previously
characterized as an interactor of ALG-2 (Missotten et al., 1999).
Recently it has been reported that ALIX plays a role in the biogenesis of intralumenal
vesicles (ILVs). The lysobisphosphatidic acid (LBPA)-mediated recruitment of ALIX
onto the endosome limiting membrane leads to the partial insertion of a hydrophobic
loop present within an exposed site of ALIX Bro1 domain into the membrane
cytoplasmic leaflet. This in turn causes local perturbations of the bilayer organization,
followed by ALIX dimerization, ESCRT-III-binding and assembly. The same
mechanism controls ILV back-fusion with the limiting membrane, either indirectly by
controlling intralumenal membrane homeostasis, or more directly by providing a
privileged site for back-fusion events within the endosome lumen (Bissig and
Gruenberg, 2014).
Proteins and lipids that transit trough endosomes, including LDL-derived cholesterol,
utilize this ILV back-fusion as an escape route from lysosomes (Falguieres et al., 2008;
Falguieres et al., 2012). Cholesterol is abundant in ILVs (Mobius et al., 2003) and, since
the mechanism of its export from endosomes to other subcellular destinations is still a
matter of debate (Ikonen, 2008; Maxfield and van Meer, 2010), several lines of
evidence indicate that the LBPA and its partner protein ALIX play a direct role in
cholesterol export (Bissig and Gruenberg, 2013).
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_______________________________________________________________4-Results
4.11 In PG cells TnBVANK1 colocalizes with ALIX positive endosomes
Using an antibody directed against ALIX (Tsuda et al., 2006), I analyzed the
distribution of this protein in yw;phm-Gal4 and phm-Gal4>TnBVank1 PGs. According
to its multifunctional activity (Odorizzi, 2006), ALIX was found widely distributed in
the cytoplasm of control PG cells (Figure 27A), and, as expected, marked some Hrspositive vesicles (Figure 27B). In the phm-Gal4>TnBVank1 PG cells the anti-ALIX
antibody detected stroke-shaped structures similar to TnBVANK1 signal (Figure 27C,
E). The immunostaining analysis performed with anti-ALIX and anti-TnBVANK1
antibodies revealed in the phm-Gal4>TnBVank1 PG cells a strong colocalization of the
two signals (Pearson’s coefficient: 0.99 ± 0.07; Figure 27D).
In addition, several of these ALIX positive stroke-shaped structures colocalized with
Hrs marked endosomes (Pearson’s coefficient: 0.95 ± 0.16), indicating that these are
modified endocytic vesicles (Figure 27F).
This strong interaction of TnBVANK1 with ALIX-containing vesicles and the altered
cholesterol distribution observed in PG are concurrent evidence that the cholesterol
route is altered. Therefore, the interaction between TnBVANK1 and endosomes
specifically affects the endosomal trafficking of sterols, likely limiting their supply to
subcellular compartments where ecdysteroid biosynthesis takes place (Gilbert and
Warren, 2005).
77
_______________________________________________________________4-Results
Figure 27. TnBVANK1 protein colocalizes with ALIX marked endosomes. Confocal images
of PG of yw;phm-Gal4 (A, B) and phm-Gal4>TnBVank1 (C-F) larvae stained for ALIX (red)
and TnBVANK1 (cyan) or Hrs (cyan). In the control PG cells ALIX (red) and Hrs (cyan) are
widely distributed in the cytoplasm and their signals partially overlap (B). In phmGal4>TnBVank1 PG cells (C-F) most of ALIX-marked vesicles have a different shape
compared to that of the controls (A, B). Immunostaining with anti-ALIX and anti-TnBVANK1
(cyan) shows a strong colocalization between TnBVANK1 signal and the ALIX stroke-shaped
vesicles (D). In phm-Gal4>TnBVank1 PG cells, most of the ALIX (red) and Hrs (cyan)
modified vesicles colocalize (F). PGs are at the same magnification and the reference scale bar
is shown in A.
78
_______________________________________________________________4-Results
4.12 ALIX knockdown in the PG cells impairs larval development and lipid
endosomal trafficking
To investigate ALIX involvement in the PG endocytic pathway, I knocked down
ALIX gene function by overexpressing a specific RNA interference transgene. I took
advantage of the UAS-ALIX-RNAi transgene containing inverted repeats of 328 bp
designed to target a region of ALIX mRNA (Dietzl et al., 2007).
I specifically induced the expression of ALIX-RNAi in the PG using the phm-Gal4
driver. All the phm-Gal4>ALIX-RNAi larvae completed the embryonic and larval
development, but, interestingly, they showed an extended third larval instar. In fact,
they reached the pupal stage four days after the yw;phm-Gal4 control larvae.
Since a prolonged larval life has also been observed when TnBVank1 was expressed in
the PG, I performed an Oil Red O staining to evaluate the potential alteration of
endocytosis due to ALIX silencing. In ALIX knockdown PG the lipid vesicular
trafficking is impaired (Figure 28B) compared to yw;phm-Gal4 control PG (Figure
28A), as showed in phm-Gal4>TnBVank1 (Figure 23C, C’).
Figure 28. ALIX silencing in PG cells alters the lipid vesicular trafficking. (A) In control
yw;phm-Gal4 there are few lipid droplets stained with Oil Red O, while in phm-Gal4>ALIXRNAi PG all cells show the increased accumulation of lipid droplets (B). Panels are at the same
magnification and the reference scale bar is shown in A.
79
_______________________________________________________________4-Results
Therefore, this result suggests that TnBVANK1 may hamper ALIX function in the
cholesterol endocytic pathway.
80
5-Discussion
81
____________________________________________________________5-Discussion
The associations between parasitic wasps and PDVs represent unique examples
of virus domestication by a cellular organism, the parasitic wasp, to manipulate the
physiology of another organism, the lepidopteran host (Bezier et al., 2009).
PDV infection contributes to a number of developmental and reproductive alterations
associated with immunosuppression and disruption of host endocrine balance (Webb et
al., 2000; Webb and Strand, 2005; Pennacchio and Strand, 2006). Relatively more
studies have addressed the host immunosuppression mechanisms, focusing on virulence
factors of the ankyrin gene family, largely shared among different taxa (Strand, 2012a).
The proteins encoded by PDV ankyrin genes show significant sequence similarity with
members of the IκB protein family involved in the control of NF-κB signaling pathways
in insects and vertebrates (Silverman and Maniatis, 2001). Since they lack the N- and Cterminal domains controlling their signal-induced and basal degradation, they are able
to bind NF-κB and prevent its entry into the nucleus to activate the transcription of
genes under κB promoters (Thoetkiattikul et al., 2005; Falabella et al., 2007; Bitra et al.,
2012).
The ankyrin gene family is one of the most widely distributed in PDVs and contains
members which are rather conserved across viral isolates associated with different wasp
species (Kroemer and Webb, 2005; Thoetkiattikul et al., 2005; Falabella et al., 2007;
Shi et al., 2008; Strand, 2012b). These genes likely originated from horizontal gene
transfer from eukaryotes, possibly the wasp itself, the host or another organism.
Consistent with this possibility, the nudiviruses, ancestors of bracoviruses (Bezier et al.,
2009), do not encode any gene showing similarity with ankyrin family members. Their
multiple acquisition and stabilization in different evolutionary lineages are clearly
indicative of the key role they play in successful parasitism. This also suggests that
ankyrin genes may be involved in multiple tasks during host parasitization, by
82
____________________________________________________________5-Discussion
influencing different physiological pathways. While an immunosuppressive function
has been demonstrated for the PDV ankyrin gene family (Thoetkiattikul et al., 2005;
Falabella et al., 2007; Bitra et al., 2012; Gueguen et al., 2013), if and how these viral
genes impact endocrine pathways or other targets has not yet been addressed.
Here, I provided experimental data that corroborate this hypothesis for TnBVank1, a
gene of the bracovirus associated with the wasp Toxoneuron nigriceps (TnBV), which
parasitizes the larval stages of the tobacco budworm, Heliothis virescens.
To better carry out my functional analyses I decided to use a well-established model
organism, which offers me a variety of experimental tools that would not be available in
H. virescens. Considering that many basic processes are evolutionarily conserved
among the insects, Drosophila melanogaster appeared to be the best choice, not only
because a plenty of molecular genetics techniques have been developed in this insect,
but also because much information is available on Drosophila endocrine system.
I showed that TnBVANK1 protein acts as a virulence factor in the prothoracic gland
disrupting ecdysone biosynthesis and thus blocking the pupa formation.
Of note, the developmental arrest at L3 induced by TnBVank1 gene expression in the
PG perfectly mimics the developmental alteration of parasitized tobacco budworm
larvae. In fact, H. virescens last instar larvae parasitized by endophagous braconid T.
nigriceps fail to attain the pupal stage, due to a parasitoid-induced alteration of the
endocrine system (Pennacchio et al., 1993; Pennacchio et al., 1997).
In phm-Gal4>TnBVank1 larvae the 20E peak, which directs the entering in the
wandering stage, is absent and the level of 20E remains extremely reduced during the
extended larval life. Moreover, the larvae expressing TnBVank1 in the PG, fed with 20E
just before the onset of the ecdysteroid peak, are able to reach the pupal stage. In light
of these data, it is reasonable to hypothesize that the developmental arrest may be due to
83
____________________________________________________________5-Discussion
low levels of 20E. In agreement with this consideration, the expression of genes
involved in ecdysone biosynthesis does not increase in the late third instar, as it occurs
in wild-type larvae (Moeller et al., 2013). These results confirm that the developmental
block induced by TnBVANK1 during the third larval instar is caused by a reduced level
of 20E in the whole body of phm-Gal4>TnBVank1 larvae.
Similarly, it has been reported that the failure to pupariate of parasitized H. virescens
last instar larvae is caused by the reduced amount of 20E produced by the depressed
biosynthetic activity of PG (Pennacchio et al., 1993).
Furthermore, the analysis of PG expressing the transgene TnBVank1 revealed that PGs
from control larvae are significantly larger than phm-Gal4>TnBVank1 PGs, but the cell
area does not show a significant reduction between phm-Gal4>TnBVank1 and control
PG cells.
The reduced gland size observed in parasitized larvae and the low basal production of
ecdysteroids (Pennacchio et al., 1997; Pennacchio et al., 1998) are fully compatible with
a general reduction of the biosynthetic activity likely induced by TnBV ank genes.
However, in naturally parasitized larvae these symptoms are also associated with a
disruption of PTTH signaling, which requires active TnBV infection of PG, where
different viral genes are expressed (Pennacchio et al., 2001; Falabella et al., 2006).
Immunostaining analyses with phalloidin, α-tubulinGFP and Dynein heavy chain
antibody show that TnBVANK1 disrupts the cytoskeletal structure of PG cells. Since
the expression of TnBVank1 in different tissues does not alter the developmental timing
and adult formation, as demonstrated by using a wide range of tissue-specific Gal4
drivers, the disruption of the cytoskeleton appears to be a PG-specific alteration.
Indeed, in a previous work it has been reported that the targeted expression of this ank
gene in Drosophila germ cells alters microtubule network function in the oocyte, as
84
____________________________________________________________5-Discussion
shown by the mislocalization of several maternal clues, without affecting the
cytoskeletal structure (Duchi et al., 2010). Therefore, I cannot exclude that the specific
effect of TnBVANK1 on the cytoskeleton of PG cells may have a negative impact on
ecdysteroidogenesis. However, the disruption of the cytoskeletal structure of these cells
may rather be a downstream consequence of the impaired steroidogenic activity.
The ecdysone biosynthesis in the PG cells is preceded by the internalization of sterols
precursors. TnBVANK1 does not affect lipid uptake, however the endocytic pathway is
impaired. In fact, stainings with Oil Red O and Filipin in the phm-Gal4>TnBVank1 PG
cells show high accumulation of lipid and sterol-rich vesicles.
The altered cell physiology and consequent accumulation of lipids and sterols may have
wide-ranging and more generalized effects on cell architecture/dynamics and survival.
In fact, the prolonged expression of TnBVank1 through phm-Gal4 during larval
development induces apoptosis of a few cells, which could account for the observed
reduction of the PG size.
In the same way, in H. virescens larvae, 120 h after parasitoid oviposition, cells start to
undergo apoptosis (Pennacchio et al., 1997).
My immunostaining analysis performed with the Rab antibodies reveals that TnBVank1
expression in PG cells causes an evident alteration of the endocytic pathway, which
culminates in a reduction of Rab7-marked endosomes. Particularly, the endosomal
system seems to be paused at the maturation of the early endosomes into late
endosomes.
During the early-to-late endosome transition, MVBs form on early endosomal
membranes and mediate transport to late endosomes. The strong colocalization of the
TnBVANK1 and Hrs immuno-detected signals suggests that this viral ankyrin protein is
associated with MVBs.
85
____________________________________________________________5-Discussion
In addition, the finding that TnBVANK1 interacts with ALIX positive vesicles, altering
their shape, strongly corroborates that in these cells the sterol trafficking is affected.
Using an RNA interference approach, ALIX knockdown in the PG prolongs the last
larval instar and interferes with sterol trafficking, as indicated by the accumulation of
lipids stained by Oil Red O. Since the results obtained by silencing ALIX mimic those
observed when TnBVank1 is expressed in the PG, it is reasonable to hypothesize that
TnBVANK1 thwart the correct functioning of ALIX in the cholesterol trafficking
endocytic pathway.
Recently, several lines of evidence indicate that in mammalian cells the lipid LBPA and
its partner ALIX play a role in controlling the cholesterol export from endosomes
(Bissig and Gruenberg, 2013). When ALIX interacts with LBPA via a mobile
hydrophobic loop and promotes the ESCRT-III filaments nucleation, it induces
perturbations of the endosomal limiting membrane. In particular, changes in membrane
symmetry and curvature help clear the intralumenal face of the bilayer of its glycocalyxlike cover. In this way, the corresponding region of the membrane is prone to serve as
the ILV docking site. The cholesterol stored into the ILVs can then leave the endosomal
compartment to move to other membrane compartments, including the endoplasmic
reticulum and the mitochondria, for ecdysone biosynthesis steps (Bissig and Gruenberg,
2014). Evidences from my in vivo studies suggest that ALIX could regulate the
vesicular trafficking with the same mechanism also in Drosophila.
A possible model explaining the TnBVANK1 function states that its expression in PG
cells blocks the cholesterol export from endosomes. In agreement with the results
reported here, I suppose that TnBVANK1 may prevent the correct interaction between
ALIX and its partners LBPA and ESCRT-III on the cytoplasmic leaflet of the
endosomes. ILVs cannot do back-fusion with the limiting membrane and thus the
86
____________________________________________________________5-Discussion
cholesterol is trapped into the MVBs (Figure 29). Finally, this block leads to insufficient
sterol supplies to reach the ecdysone level necessary to complete development.
Figure 29. Proposed model of TnBVANK1 action in MVBs of PG cells. In phmGal4>TnBVank1 TnBVANK1 may avoid the interaction between ALIX, LBPA and ESCRT-III
on the cytoplasmic leaflet of the MVBs. ILVs cannot do back-fusion with the limiting
membrane and thus the LDL-derived cholesterol is trapped into the MVBs. Modified from
(Bissig and Gruenberg, 2014).
The high similarity of the recorded phenotypes represents a solid background from
which to start the design of specific experiments on the natural host. Indeed, the results
reported here set the stage for specific in vivo studies, that will help address the
respective roles of different PG-directed TnBV genes in the suppression of
ecdysteroidogenesis in parasitized host larvae.
87
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